pmid stringlengths 8 8 | pmcid stringlengths 8 11 ⌀ | source stringclasses 2
values | rank int64 1 9.78k | sections unknown | tokens int64 3 46.7k |
|---|---|---|---|---|---|
40104552 | PMC11914766 | pmc | 5,963 | {
"abstract": "Highlights • Soil and its microbial communities play a pivotal role in nutrient cycling, plant growth, and overall ecosystem health. • Over time, the co-evolution of plant-microbe relationships has fostered an interdependence that is central to the microbial loop, significantly contributing to ecosystem functionality. • The ecological and biological dimensions of food production necessitate the adoption of sustainable practices that preserve microbial diversity and promote a holistic management approach to enhance agricultural productivity. • Co-occurrence analysis synthesizes insights across the sections and validates the discussed topics, reinforcing the need for comprehensive scientific understanding to guide effective agricultural practices and policy-making."
} | 193 |
35072828 | PMC8787007 | pmc | 5,965 | {
"abstract": "Background Although microbial fuel cells (MFCs) represent a promising technology for capturing renewable energy from wastewater, their scaling-up is significantly limited by a slow-rate cathodic oxygen reduction reaction (ORR) and the development of a resilient anodic microbial community. In this study, mixed transition metal oxides of nickel and copper (Ni and Cu), supported on a graphene (G) (NiO–CuO/G) electrocatalyst, were synthesized and tested as a cost-effective cathode for ORR in MFCs. Electrochemical measurements of electrocatalyst were conducted using a rotating disk electrode (RDE) and linear sweep voltammetry (LSV) in a neutral electrolyte, and compared with a benchmark Pt/C catalyst. Furthermore, the long-term performance of the as-synthesized electrocatalyst was evaluated in a single-chamber MFC by measuring organic matter removal and polarization behavior. The successful enrichment of electroactive biofilm was also monitored using transmission electron microscopy and the Vitek2 compact system technique. Results When compared with the benchmark platinum cathode, the NiO–CuO/G electrocatalyst exhibited high selectivity toward ORR. The rotating disk electrode (RDE) experiments reveal that ORR proceeds via a 4-electron ORR mechanism. Furthermore, the NiO–CuO/G electrocatalyst also exhibited a high power density of 21.25 mW m −2 in an air-cathode MFC, which was slightly lower than that of Pt/C-based MFC (i.e., 50.4 mW m −2 ). Biochemical characterization of the most abundant bacteria on anodic biofilms identified four genera (i.e., Escherichia coli , Shewanella putrefaciens , Bacillus cereus , and Bacillus Thuringiensis/mycoides ) that belonged to Gammaproteobacteria , and Firmicutes phyla. Conclusions This study demonstrates that the NiO–CuO/G cathode had an enhanced electrocatalytic activity toward ORR in a pH-neutral solution. This novel mixed transition metal oxide electrocatalyst could replace expensive Pt-based catalysts for MFC applications. Supplementary Information The online version contains supplementary material available at 10.1186/s43141-021-00292-2.",
"conclusion": "Conclusions To the best of our knowledge, it was the first time to use this combination of mixed metal oxides (NiO–CuO/G) for application in MFC as a cathode electrocatalyst. The successful preparation of NiO–CuO/G electrocatalyst was confirmed by XRD, TEM, SEM, and EDX analysis. The electrochemical characterization showed high selectivity and electrocatalytic activity of the electrocatalyst towards the ORR that follows the four-electron pathway. The efficiency of NiO–CuO/G in MFCs yielded a maximum PD of 21.3 mW m −2 with a C E of 25 ± 0.71%. These results were slightly lower than Pt/C based MFCs (PD = 50.4 mW m −2 and C E = 35 ± 0.69%). The enhanced electrocatalytic activity of NiO–CuO/G may be mainly due to its high surface area and synergistic effect between NiO/CuO and graphene. These synergistic effects provide NiO/CuO surface with large amounts of active sites, resulting in high stability of the electrocatalyst and enhanced electrical conductivity. This enhanced the performance of MFCs. Both SEM and TEM analysis of anodic biofilm showed the rod-shaped structure of electroactive microorganisms, confirming that the generated electricity was due to the formed electroactive biofilm on the surface of the anodic electrode. In addition, the biochemical characterization of the anodic communities reveals a possible pathway for the isolation of EAB via anaerobic enrichment and was primarily anticipated as a tool for selecting EAB consortia. This research provides new perspectives into discovering effective non-precious mixed metal oxides (NiO–CuO/G) cathode electrocatalyst as a replacement for noble and very costly Pt/C for practical applications of MFCs. The future work will be relying on exploring different combinations of transition metal oxides as ORR electrocatalysts, such as nickel along with cobalt or manganese oxides. The performance of these electrocatalysts will be evaluated using different physical and electrochemical techniques. Moreover, their applications in MFC with different constructions and operating conditions will be performed.",
"discussion": "Discussion In this study, the NiO–CuO/G electrocatalyst was developed as a promising cathode catalyst for MFC applications. Successful catalyst preparation was confirmed using XRD, TEM, SEM, and EDX analyses. First, XRD data revealed that the crystalline nature of the graphite framework supported the electrocatalyst [ 24 ]. Furthermore, the C (002) peak became softer as the metal particles on the G fill-up the diffraction toward the composites, leading to a G peak reduction. The 2 θ values indicated the presence of crystal planes (111), (200), (220), and (311) of NiO and CuO, which were assigned to (− 111), (200), (− 202), (202), and (220) planes. Thus, mixed metal oxides could exist as NiO and CuO, suggesting the effective synthesis of a NiO/CuO composite [ 52 , 53 ]. The representative peaks (Fig. 1 ) confirmed the with successful deposition of NiO and CuO on G [ 54 ]. This deposition may have been due to G’s elevated surface area and conductivity with available active sites, which may have enhanced the bioelectrochemical efficiency of NiO–ORR [ 28 ]. Second, TEM observations agreed with those by Li et al. [ 55 ], who prepared a G–Co/Ni composite on carbon cloth electrodes(G–Co/Ni–CC). These researchers reported large amounts of small-sized Co/Ni composites randomly deposited on the surface of crumpled G sheets. Third, from SEM and EDX analyses, NiO and CuO were effectively precipitated on G using the in situ preparation method, with percentages close to the nominal ratio of C, O, Ni, and Cu. These findings indicated that NiO and CuO was successfully deposited on the G surface. The electrochemical characterization of as-synthesized electrocatalyst when compared with the Pt toward ORR in neutral media was evaluated by LSV. These data suggested that NiO–CuO/G considerably enhanced redox reaction performance and exhibited an electrochemical activity toward ORR comparable with Pt. The current density increased with an increase in rotation speed and a decrease in potential scan to more negative values. This was explained by the transmission of steady streams of the bulk solution to the electrode surface during high rotations, whereas the bulk solution that is far from the electrode surface remains well stirred by the convection and the shortened diffusion distance at subjected speeds [ 29 , 56 ]. Moreover, kinetic analyses (based on the K-L relationship) revealed that the four-electron pathway directly to water mainly dominates the ORR in Pt/C similar to ORR catalyzed by NiO–CuO/GG according to the following equation:\n 4 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathrm{O}}_{2}+4{\\mathrm{H}}^{+}+4{\\mathrm{e}}^{\\hbox{-}}\\to 2{\\mathrm{H}}_{2}\\mathrm{O}$$\\end{document} O 2 + 4 H + + 4 e - → 2 H 2 O These results demonstrated an improved electron transfer for the NiO–CuO/G electrode. The distribution of electron transfer numbers confirmed that NiO–CuO/G improved the ORR and improved the performance of the cathode in MFCs in neutral media. These data agreed with a previous study in which silver-tungsten carbide (Ag–WC)/C nano-hybrids showed a comparable ORR efficiency to Pt/C in MFCs [ 35 ]. This high ORR activity might have been due to synergistic effects between carbon, WC, and Ag nanoparticles. The NiO–CuO/G contributed to an efficient catalytic ORR activity. Therefore, catalytic activity toward ORR was identified from the combined impact of NiO and CuO loaded onto G. Furthermore, the elevated ORR performance of NiO–CuO was due to the high catalytic surface area of NiO–CuO particles, there by facilitating high deposition rates of the catalyst onto G surfaces and the high porosity of the composites. These results provided evidence on the use of cheaper Pt-free mixed metal oxides as electrocatalysts in MFCs, without significantly impeding performance. The evaluation of electrocatalyst performance in MFCs showed that maximum OCV values were correlated with an increase in reaction rates, thereby allowing the adsorption and diffusion of higher amounts of O 2 onto the electrocatalyst surface [ 57 ]. The performance of the NiO–CuO/G cathode in MFCs was comparable to Pt/C during the biofilm acclimation period (62 days). NiO–CuO/G-based MFCs had a maximum PD of 21.3 mW m −2 and C E of 25% ± 0.71%, which is somewhat close to Pt/C-based MFCs (e.g., PD = 50.4 mW m −2 and C E = 35 ± 0.69). These data suggested that C E was determined mainly by cathode variations, but they might have been attributable to the ideal properties of the NiO–CuO/G electrocatalyst. The formation of the biofilm is dependent on the intensive development of the bacterial cells at a high COD removal value. It can be presumed that the higher COD removal is directly correlated to the enhanced substrate utilization and the comparatively higher performance with improved power output [ 24 , 28 ]. The high surface area of G’s uniform distribution and dispersion of the prepared catalyst led to a higher voltage. This led to a higher current output of the system, with higher power output. Thus, the overall reaction of the NiO–CuO/G, with a high COD removal efficiency, revealed that NiO–CuO/G cathode may be efficiently used as an electrocatalyst for MFC applications. Surface analyses of anodic biofilms using SEM and TEM indicate that bacterial cells were rod-shaped structures indicative of electroactive microorganisms and thus confirming that the generated electricity was due to electroactive biofilms on the surface of anodic electrodes. In addition, the biochemical characterization of anodic bacterial communities revealed a possible mechanism for the isolation of electrochemically active bacteria (EAB). These analyses indicate that the anode itself is supposed to be a pathway through an anaerobic enrichment of anodic biofilm. The approach considers the fundamental properties of living organisms to absorb respiratory energy via electrons. Bacteria use this energy as an alternative to direct respiration in the absence of an electron acceptor. The literature survey yielded that non-precious Ni-based electrocatalysts generally exhibited good catalytic activity and stability comparable to Pt/C cathode in MFCs (Table 2 ). Table 2 A comparative study of the performance of MFC between using different nickel-based electrocatalyst Cathode catalyst Anode material Cathode material Substrate MFC configuration Microorganism Open circuit potential (mV) closed circuit voltage (mV) PD max (mW.m −2 ) Percentage to Pt cathode (%) Ref. NiO–CuO/G Carbon felt Carbon cloth Sodium acetate Air cathode Activated sludge 654 541 21.25 42.16 This study Nickel nanoparticles on reduced graphene oxide Graphite brush Carbon cloth Sodium acetate Air cathode Anaerobic digester sludge 602 136.8 581 26.4 (Valipour et al. 2016) [ 5 ] Naphthalocyanine on carbon black (NPc/C) Carbon paper Carbon paper Wastewater Double chamber Anaerobic digester sludge 602 168 29.7 36.53 (Rae et al. 2011) [ 58 ] Nickel oxide and carbon nanotube composite (NiO/CNT) Carbon felts Carbon cloth Glucose Air cathode Acclimated sludge from methane-generating pond 772 380 670 N/A a (Huang et al. 2015) [ 29 ] Pt-Ni alloy Nano particles on Carboxyl multi-wall carbon nanotubes (Pt-Ni/MWNT) Carbon cloth Carbon cloth Glucose Air cathode Pre-domesticated bacteria from another double chamber MFC 740 570 1.22 86.8 (Yan et al. 2012) [ 16 ] Ni-tetra sulfonated phthalocyanine Stainless steel foam was modified with rGO Carbon felt Sodium acetate Double chamber A mixture of compost garden’s leachate 24.8 N/A a (Champavert et al. 2015) [ 32 ] Graphene/nickel hybrids Graphite plate Graphite plate Waste water Dual chamber Wastewater 34 N/A a (Kartick et al. 2016) [ 31 ] Nickel nanoparticles Carbon paper Carbon paper Glucose Dual chamber Palm oil mill effluent anaerobic sludge 751.8 94.4 78.15 (Ghasemi et al. 2013) [ 28 ] CNT carbon nanotube, MWNT multi-wall carbon nanotubes a \n N/A not available Ni-based electrocatalysts could be used as a cathode catalyst in the MFC as evidenced by the high PD in MFCs. The as-synthesized electrocatalyst was found to be more efficient than those reported by Liu and Vipulanandan [ 30 ]. These authors used Ni nanoparticles for a two-chamber MFC application that generated a PD of 0.07 mW m −2 . In another study, Champavert et al. [ 32 ] fabricated a carbon felt cathode modified with poly Ni (II) tetra sulfophthalocyanine for a dual-chamber MFC. This generated a PD of 21.6 mW m −2 , which was comparable to our results (21.3 mW m −2 ). This difference in PD may have been due to several factors, including operational conditions, MFC design, anode type and surface area, differences in bacterial communities, and differences in supporting material for the electrocatalyst and its projected surface area. Our research provides new perspectives for effective non-precious mixed metal oxide (NiO–CuO/G) cathode electrocatalysts as replacements for noble and costly Pt/C for practical MFC applications. The enhanced electrocatalytic activity of NiO–CuO/G might have been primarily due to its high surface area and synergistic effects between NiO/CuO and G. These synergistic effects provided NiO/CuO surfaces with high quantities of active sites, thereby confirming electrocatalyst stability, electrical conductivity, and enhanced MFC performance."
} | 3,436 |
30122051 | PMC6291805 | pmc | 5,968 | {
"abstract": "DNA\nnanotechnology provides a versatile toolbox for creating custom\nand accurate shapes that can serve as versatile templates for nanopatterning.\nThese DNA templates can be used as molecular-scale precision tools\nin, for example, biosensing, nanometrology, and super-resolution imaging,\nand biocompatible scaffolds for arranging other nano-objects, for\nexample, for drug delivery applications and molecular electronics.\nRecently, increasing attention has been paid to their potent use in\nnanophotonics since these modular templates allow a wide range of\nplasmonic and photonic ensembles ranging from DNA-directed nanoparticle\nand fluorophore arrays to entirely metallic nanostructures. This Feature\nArticle focuses on the DNA-origami-based nanophotonics and plasmonics—especially\non the methods that take advantage of various substrates and interfaces\nfor the foreseen applications.",
"conclusion": "Conclusions Although there are a plethora of examples of plasmonic structures\nthat are assembled using DNA, 46 , 48 , 49 so far only a few examples of DNA-based plasmonic interfaces or\nprototypes and approaches aiming toward DNA-assisted metasurfaces\nhave been reported. However, despite all the challenges ahead, the\nfuture seems promising. As the cost of a single gram of mass-produced\nDNA origami has been reduced to 200 dollars, 10 this means that covering a square meter of the substrate would cost\nonly approximately 20 cents. 106 This kind\nof vast area patterning may become possible by employing the presented\nmethods to organize single DNA structures at interfaces or in 3D. 91 Nevertheless, another route to achieve higher-order\nsystems also exists, since approaches to create larger and larger\nDNA origami have recently been reported. 15 , 16 This progress may lead to mass production of plasmonic DNA nanoantennas\nfor sensing and intriguing optical metasurfaces consisting of miniature\nlight scatterers. 110 , 111 In general, using DNA\norigami as a bridge between bottom-up and\ntop-down fabrication methods may help to go beyond the resolution\nlimit of conventional lithography and solve the challenges of nanofabrication.\nThis way it would be possible to achieve a whole class of novel hybrid\nmaterials with tunable optical and electronic properties. 107 , 111 In addition to providing alternative approaches for solid-state\nnanofabrication, wet chemistry and structural DNA nanotechnology meet\nat the interface where DNA structures can be used in the fabrication\nof inorganic nanoparticles. 22 , 53 , 112 − 114 Integrating these components into larger\ndevices remains a challenge, but by merging the subfields with the\nhelp of DNA nanotechnology, we believe that the current achievements\npresent just the beginning of a flourishing era of DNA-origami-based\nnanophotonics.",
"introduction": "Introduction The\nrapid evolution of structural DNA nanotechnology 1 − 5 along with advanced and automated techniques, computer-aided\ndesign\nsoftware, powerful simulation tools, and reduced cost of synthesis 6 − 10 have enabled the effortless fabrication of custom DNA nanoarchitectures\nfor many applications in materials science and bio-oriented research. 3 , 11 , 12 Examples of using versatile\nDNA shapes—especially DNA origami 11 , 13 − 16 —have been reported, for example, in molecular electronics, 17 − 22 super-resolution imaging, 23 − 26 and drug delivery. 27 − 31 These customized DNA objects can also find use as\nmolecular-scale diagnostic tools, 32 dynamic\ndevices, 33 − 37 and templates for controlling chemical reactions, 38 − 40 and importantly,\nthese structures are inherently biocompatible and readily modifiable\nfor application-specific purposes. 28 , 30 , 41 − 45 These examples are enabled by virtue of the sub-nanometer structural\naccuracy of DNA origami and nanometer-scale patterning resolution\nof multiple molecular components. 11 , 13 − 16 In general, the DNA origami technique provides a straightforward\nroute from the target shape to a functional product via a self-assembly\nprocess. In a one-pot assembly, one can create a very large number\nof customized and well-defined DNA origami nanostructures ranging\nfrom nano- to micrometer size 15 and from\nmega- to gigadalton scale 16 for various\nuser-defined tasks and implementations. In addition to these\nexamples, DNA nanostructures can be used as\nprecise templates for arranging metal nanoparticles and creating plasmonic\nassemblies and nanophotonic devices. 46 − 49 Like in all of the other above-mentioned\nexamples, building nanophotonic structures using DNA is also based\non taking advantage of the programmability, modularity, and high addressability\nof the DNA nano-objects. Arguably, there are several benefits of integrating\nthe plasmonic DNA nanostructures with interfaces; for example, substrate-based\nmethods are readily compatible with conventional top-down lithography\nmethods, and they also enable characterization of individual nanodevices. 50 − 53 Moreover, DNA lattice-based approaches can be improved by taking\nadvantage of DNA structure diffusion on substrates. 54 , 55 Importantly, anchoring of the nanostructures to interfaces facilitates\ncoupling of nanodevices with outer circuitry, 18 diagnostic and sensing tools, 32 and fabrication\nof bioinspired substrates and metasurfaces. 56 In this Feature Article, we summarize the key aspects of DNA\norigami\nplasmonics and provide an overview of placement and arrangement methods\nfor self-assembled DNA nanostructures. Finally, we combine the methodology\nof these two sections and discuss the recent progress of DNA-origami-based\nnanophotonics at interfaces. DNA-Nanostructure-Based Nanophotonics and\nPlasmonics: An Overview The highly addressable DNA origami\nprovides an excellent platform\nto organize nanophotonic components into systems with emerging optical\nproperties because of its nanometer-scale precision control. The most\ncommon photonic objects arranged with DNA origami are metallic nanoparticles\n(NPs) and fluorophores. When NPs interact with light, their conduction-band\nelectrons can enter a collective oscillation mode, that is, a so-called\nlocalized surface plasmon resonance (LSPR). The plasmon oscillation\nis highly sensitive to the polarizability of the particle; in other\nwords, the composition, geometry, and surrounding media of the NP\nall have a significant influence on the resonance. In addition, when\ntwo NPs are in close proximity, their plasmon oscillations can couple\nwith each other, resulting in intriguing optical properties and remarkable\nelectromagnetic field enhancement. This coupling is strongly dependent\non the spatial arrangement of NPs. For the tailoring of the optical\nresponse of the NP systems, techniques to position specific NPs with\naccurate spatial configuration are of vital importance. To date, DNA\norigami is one of the most promising candidates to tackle this issue\nwith its sub-nanometer- to nanometer-scale positioning precision for\nmolecules and nanoparticles. 57 , 58 In addition to this\nextreme positioning resolution, the accessibility and absolute incorporation\nefficiency of the single strands in DNA origami can reach 95%. 59 In 2010, the first DNA-origami-guided\narrangement of spherical gold NPs (AuNPs) was demonstrated. 60 The desired NP arrangement was achieved via\ncoating AuNPs with oligonucleotides and further via their hybridization\nwith complementary strands extended from the specific positions of\norigami. Later on, a plethora of NP assemblies with customized optical\nproperties have been developed. 46 , 48 , 49 A representative example of such a rationally designed plasmonic\nDNA device is a chiral plasmonic structure assembled from a rodlike\nDNA origami and nine AuNPs that go around the DNA rod in a helical\nfashion ( Figure 1 a). 61 These chiral plasmonic nanostructures exhibited\nsignificant circular dichroism (CD) in the visible range, and their\nCD responses could be tuned by altering the NP size and the helical\nhandedness. Spherical AuNPs can also be arranged into a ring conformation\nto obtain tunable electric and magnetic plasmon resonances at the\nvisible frequencies. 62 In addition to AuNPs,\nspherical silver NPs (AgNPs) have also been organized by DNA origami. 63 − 65 Figure 1 (a)\nLeft: DNA-origami-based left- and right-handed chiral plasmonic\nnanoassemblies that show strong circular dichroism. 61 Right: Transmission electron micrograph of stacked assembly.\n(b) Dynamic chiral metamolecule that is sensitive to the pH change\nof solution. 36 (c) AuNP dimer with defined\ndistance assembled on a DNA origami platform. 78 (d) Heterotrimer assembly with a AgNP between two AuNPs on a DNA\norigami beam. 81 (e) Bar-shaped DNA origami\nwith docker strands on sides to capture transient imager strands for\nsuper-resolution imaging. 25 (f) DNA origami\nwith multiple programmable fluorescent dye binding sites acts as a\nmetafluorophore. 84 Part a is reprinted\nwith permission from ref ( 61 ). Copyright 2012 Springer Nature. Part b is reprinted with\npermission from ref ( 36 ). Part c is reprinted with permission from ref ( 78 ). Part d is reprinted with\npermission from ref ( 81 ). Copyright 2017 Springer Nature. Part e is reprinted with permission\nfrom ref ( 25 ). Copyright\n2014 Springer Nature. Part f is reprinted with permission from ref ( 84 ). Since the LSPR is sensitive to the geometry of NPs, metallic\nNPs\nwith shapes other than a sphere could offer more diverse optical properties\nand therefore further facilitate the design of nanophotonic devices.\nDNA origami conjugates with gold nanorods (AuNRs), 34 − 36 , 66 , 67 gold nanotriangles\n(AuNTs), 68 or hybrid assemblies of particles\nwith different geometries 69 , 70 have also been explored. In addition to static nanophotonics, dynamic and reconfigurable\nplasmonic DNA devices have been developed. They are considered dynamic\nsince the geometric configuration of such devices could be changed\nand switched after assembly. For example, by attaching two AuNRs to\nDNA origami beams connected via a single Holliday junction, a metamolecule\nwith different rotational states—and therefore different CD\nsignal states—can be formed. The reconfiguration of the states\ncan be induced by the introduction of DNA displacement strands, 34 by light, 35 or by\nchanging solution pH (see Figure 1 b). 36 In addition to these,\nTurek et al. have demonstrated how thermoresponsive polymer-equipped\nDNA tweezers could be used as an actuator for surface-enhanced fluorescence. 71 Instead of reconfiguring the whole metamolecule, the AuNR itself\ncan also “walk” between different sites on a DNA origami\nby fueling the system with displacement strands 67 using a similar strategy as originally introduced by Yurke\net al. 72 Significant effort has been\nput into developing techniques to position\nNPs in extremely close proximity to form hot-spots for the field enhancement,\nwhich is essential for surface-enhanced Raman spectroscopy (SERS)\nand fluorescence enhancement (FE). To achieve the nanometer-scale\ndistance, different strategies have been employed. For example, “seed-NPs”\nthat are attached to DNA origami could be grown larger by chemical\nmethods to shorten their distance. 19 , 73 , 74 Upon conjugation of NP dimers on different sides\nof DNA origami, the DNA helices between NPs can act as binding sites\nand as a spacer of a few nanometer thickness. 75 − 77 Rationally\ndesigned DNA origami structures could also be used to dock NP dimers\nwith a gap of only a few nanometers, as shown in the work of Thacker\net al. (see Figure 1 c) 78 and Roller et al. 79 In these works, the local field was enhanced by several\norders of magnitude and demonstrated by SERS or FE characterization. To fully take advantage of the addressability and versatility of\nDNA origami, plasmonic systems with different metal compositions have\nalso been assembled. These heteroparticle assemblies 80 , 81 have shown optical modes that are challenging to obtain with any\nother method. For example, a nondissipative and ultrafast plasmon\npassage has been observed in a heterotrimer system consisting of both\na AuNP and AgNP arranged on top of DNA origami (see Figure 1 d). 81 Fluorophores form another group of nanophotonic components\nthat\nare widely combined with DNA nanostructures. DNA origami can work\nas a nanoadapter to enable manipulation of individual or a few fluorescent\nmolecules. In addition to the well-known Förster resonance\nenergy transfer (FRET) studies, 49 , 82 DNA origami structures\nequipped with dye-labeled oligonucleotides have been used in DNA-point\naccumulation for imaging in nanoscale topography (DNA-PAINT) in super-resolution\nmicroscopy (see Figure 1 e), 24 , 25 , 83 and as a nanoruler\nfor spatial calibration. 23 Upon combination\nof various different fluorescence dyes on a single rectangular DNA\norigami, it is possible to assemble a so-called metafluorophore (see Figure 1 f). 84 DNA Origami Placement at Interfaces As already explained,\nthere are a number of advantages in anchoring the DNA-based devices\nto the substrates or incorporating them into larger systems for nanophotonics.\nThis section deals with the approaches suitable for assembling DNA\nstructures at the interfaces. DNA nanostructures can be immobilized\nonto substrates merely by electrostatic interactions, and one of the\nmost common methods is adhesion to mica with the presence of magnesium\nions. 85 Upon adjustment of the amount of\ncations of the deposition buffer, DNA nanostructures can be placed,\nfor example, on silicon and silicon oxide. 50 , 51 , 53 Interestingly, monovalent cations can be\nused for surface-assisted assembly of higher-order structures. 54 , 55 Aghebat Rafat et al. used cross-tile structures ( Figure 2 a), and created 2D lattices\nby controlling the diffusion of the structures on a mica substrate\nwith monovalent cations. 54 The cross-tiles\nwere similar to ones designed by Liu et al., 86 except that the tiles were “twist-corrected”, (i.e.,\nthe undesired twist caused by the square-lattice design 87 was removed), and they formed the close-packed\ncrystalline structures via blunt-end stacking interactions 88 , 89 instead of sticky-end cohesion. A similar approach of lattice organization\non the substrate has also been demonstrated for a rectangular origami 55 and an origami triangle 13 that has a void in the middle of the structure. The triangles form\na close-packed lattice that could be further used as a removable mask\nfor protein patterning of the substrate through the openings of the\ntriangle layer. 90 Moreover, blunt-end stacking\nof DNA origami objects could be applied to arranging DNA origami in\n3D lattices. Recently, Zhang et al. 91 used\nthree-dimensional and rigid origami tensegrity triangles—similar\nto simpler tensegrity triangles in the very first 3D DNA crystal by\nZheng et al. 92 —to form a 3D lattice\nwith voids via blunt-end stacking. They also demonstrated the addressability\nof the origami lattice by attaching gold particles to the lattice\nvoids, thus creating a hybrid DNA–gold nanoparticle superlattice. Figure 2 (a) AFM\nimage of DNA origami cross-tiles assembled into a lattice\nconfiguration by cation-induced diffusion. 54 (b) Lipid-bilayer-facilitated 2D-lattice formation from DNA origami\ncross-tiles. 93 (c) Triangular DNA origami\nis covalently immobilized to binding sites patterned on a silicon\nsubstrate by lithography. 50 (d) Dielectrophoretic\ntrapping of 3D DNA origami between lithographically fabricated gold\nnanoelectrodes. 18 (e) DNA origami equipped\nwith a AuNP is selectively attached to a gold pattern on a silicon\nsubstrate. 101 (f) Large-scale spray-deposition\nof DNA origami nanostructures to the selected substrate through a\nmask. 103 Part a is reprinted with permission\nfrom ref ( 54 ). Copyright\n2014 John Wiley and Sons. Part b is reprinted with permission from\nref ( 93 ). Part c is\nreprinted with permission from ref ( 50 ). Copyright 2014 American Chemical Society. Part\nd is reprinted with permission from ref ( 18 ). Copyright 2015 John Wiley and Sons. Part e\nis reprinted with permission from ref ( 101 ). Copyright 2009 John Wiley and Sons. Part f\nis reprinted with permission from ref ( 103 ). In addition to mica substrates, lipid bilayers can also be\nemployed\nin the organization of DNA origami lattices and other higher-order\nassemblies 93 − 95 as seen in Figure 2 b. The lattice formation process can be controlled\nusing cholesterol-modified DNA origami and connector staples 94 or different lipid membrane phases (liquid-disordered\nor solid-ordered). 95 Moreover, the growth\nof arrays can be triggered by adjusting monovalent cation concentration\nas well as the concentration of deposited origami structures. 95 One approach to pattern substrates with\nthe resolution beyond conventional\nlithography techniques is to combine top-down approaches with the\nhigh addressability of DNA origami. Kershner et al. 96 fabricated arrays of DNA origami triangles by attaching\norigami to lithographically revealed precise triangular areas of silicon\noxide in a hexamethyldisilazane (HDMS) film. To demonstrate the potential\nof the method in high-resolution nanoscale patterning, Hung et al. 97 attached AuNPs to the corners of the origami\ntriangle and organized them to the lithographically confined wells. Later on, Gopinath & Rothemund 50 optimized the assembly of DNA origami triangle nanoarrays ( Figure 2 c) on silicon and\nsilicon oxide substrates. They reported how DNA origami triangles\ncould be covalently coupled to the lithographically patterned wells\nwith high yields using the isourea bond (as shown in Figure 2 c) or amide bond for linking.\nAttaching DNA origami to silicon or silicon oxide usually requires\ndeposition buffers with high magnesium content, but by employing covalent\nlinking instead of simple electrostatic bonds between DNA origami\nand the substrate, a much wider magnesium concentration range could\nbe used. Recently, Brassat et al. 98 further\nstudied DNA origami adsorption onto a silicon oxide surface in nanohole\narrays by varying Mg and DNA origami concentration, buffer strength,\nadsorption time, and nanohole size. They observed that buffer strength\nplays a critical role in origami deposition. In addition to\nfabricating origami-shaped wells in the films, one\ncan also use other lithographic features for directing DNA origami\nto predefined locations with targeted geometries. One example is to\ntake advantage of the (directional) polarizability of DNA origami\nand guide the structure by electromagnetic fields. Kuzyk et al. 99 and Shen et al. 18 have demonstrated how dielectrophoresis (DEP) can be used to trap\nvarious 2D and 3D DNA origami shapes between gold nanoelectrodes.\nBy applying ac voltage to the nanoelectrodes, one can create a highly\nlocalized electric field that traps DNA origami in the electrode gap\n(see Figure 2 d). The\nproper immobilization (and coupling to outer electrical circuitry)\ncan be ensured through the covalent gold–sulfur bond by incorporating\nthiolated strands into DNA origami. Nevertheless, DEP could also allow\ntransfer of the achieved trapping geometries to the chosen electrodeless\nsubstrate by the imprint technique. 100 Moreover, Gerdon et al. 101 used lithographically\npatterned gold patches functionalized with 11-mercaptoundecanoic acid\n(MUA) to selectively immobilize DNA origami (see Figure 2 e). The carboxylic acid groups\nof the functionalized layer chelate magnesium ions in the deposition\nbuffer, and thus, a salt bridge is formed between a negatively charged\nDNA origami and the positively charged Mg ions. However, compared\nto other lithographically achieved patterning methods, this technique\nlacks the control over spatial orientation of the delivered DNA origami.\nTo achieve directionality and to align the structures in a controllable\nway, Ding et al. 102 showed that small gold\nislands—patterned by e-beam lithography in different geometries—could\nbe connected by thiol-modified DNA origami nanotubes. The nanotubes\nhad exactly matching length with the distance of the neighboring gold\nislands. To achieve large-scale deposition, Linko et al. 103 developed a spray-coating technique ( Figure 2 f), which allows\nstraightforward and efficient\ndelivery of DNA origami to the substrate without a need of chemical\ntreatments or washing steps. However, the majority of salt ions has\nto be removed from the origami solution 45 before deposition. They demonstrated the feasibility of the method\nusing various 2D and 3D origami structures, removed the residual salt\nby spin-filtering, 45 and spray-coated large\nareas of glass and silicon substrates. Although one can control the\ncoating procedure at large scales using mechanical masks, the structures\nare randomly oriented on the deposited areas. However, this technique\nmight become compatible with other methods described in this section. DNA Origami Plasmonics and Nanophotonics at Interfaces By\ncombining the DNA-based nanophotonic devices and different techniques\nto arrange DNA structures on interfaces described in the previous\nsection, one can achieve plasmonic substrates with tailored optical\nproperties. One goal in this direction would be to create metasurfaces\nusing ordered assemblies, but nevertheless, the individual DNA-based\nobjects and their properties should first be characterized. To probe single plasmonic devices, they need to be deposited onto\na substrate after their self-assembly in buffer solution. In many\nreported experimental setups, 76 − 78 plasmonic structures were immobilized\nvia electrostatic interactions on a charged substrate (see the previous\nsection). Another commonly used scheme is to take advantage of the\nstrong interaction between biotin and avidin. As an example, Acuna\net al. 75 immobilized a rodlike DNA origami\nequipped with a AuNP dimer and a single fluorescent molecule in the\ngap to the substrate by biotin–neutravidin linking (see Figure 3 a). The binding and\nunbinding events of short DNA strands, as well as the conformational\ndynamics of a Holliday junction in the hot-spot, were visualized in\na form of enhanced fluorescence signals in real-time. Recently, a\nsimilar system has also been used to characterize single peridinin–chlorophyll a –protein complex. 65 Figure 3 (a) Gold\nnanoparticles and a rodlike DNA origami form a nanoantenna\nthat is attached to the substrate via biotin–neutravidin interaction. 75 (b) Chiral plasmonic nanostructures (similar\nto Figure 1 a) arranged\nonto a substrate to achieve a switchable and directional circular\ndichroism (CD) effect. 104 (c) DNA origami\ntriangles equipped with fluorophores can be precisely placed into\nan optical cavity. 51 The number of triangles\nand their position can be controlled and thus the fluorescence of\nthe “pixel” (inset) in a large array of cavities. (d)\nDNA origami is used as a nanoadapter to place individual fluorescent\nmolecules in lithographically fabricated metallic zero-mode waveguide. 107 (e) DNA-assisted lithography (DALI). 53 DNA origami (top panel) is deposited on a silicon\nchip (middle panel), and its shape is transferred into a metallic\nstructure on a transparent substrate (bottom panel). Part a is reprinted\nwith permission from ref ( 75 ). Copyright 2012 American Association for the Advancement\nof Science. Part b is reprinted with permission from ref ( 104 ). Part c is reprinted\nwith permission from ref ( 51 ). Copyright 2016 Springer Nature. Part d is reprinted with\npermission from ref ( 107 ). Copyright 2014 American Chemical Society. Part e is reprinted with\npermission from ref ( 53 ). The chiral plasmonic structures\nsimilar to the ones designed by\nKuzyk et al. 61 can also be immobilized\nby means of biotin–avidin interaction. 104 Interestingly, upon anchoring of the structures on the\nsubstrate, their CD responses became switchable. When the sample was\nimmersed in buffer, most of the structures were in an upstanding position,\nbut when the buffer was dried, the structures lay horizontally on\nthe surface. Their different relative orientations with respect to\nthe exciting circularly polarized light could induce a change in the\nCD signal (see Figure 3 b). Another benefit for depositing DNA plasmonic structures\non a surface\nis that they can be combined with materials with which it would be\notherwise challenging to assemble in the solution phase. For example,\nan origami–AuNP dimer–graphene hybrid structure was\nformed by exfoliating a single layer of graphene on top of an immobilized\nAuNP nanoantenna. 105 Such a hybrid system\nhas demonstrated superior SERS performance compared to individual\ncomponents. Along the lines of using DNA origami as nanoadapters\nto position\nnanophotonic components beyond the accuracy of conventional lithography, 50 , 96 , 97 , 106 Gopinath et al. recently demonstrated a reliable and controllable\ncoupling of molecular emitters to photonic crystal cavities (PCCs)\n(see Figure 3 c). 51 Because of the high addressability of DNA origami,\nthe location of the dye molecule was sufficiently precise to enable\nvisualization of the local density of states within PCCs with a resolution\nof about 1 / 10 of a wavelength. Moreover, the\nintensity of the cavity emission could be digitally varied by changing\nthe number of binding sites within a single cavity. Taking the high\nmodularity of DNA origami into consideration, a great number of hybrid\nnanophotonic applications could be realized with this system. In addition to relying just on the pattern with the same outline\nas DNA origami, circular openings in a metallic film can also host\nDNA origami adapters with size-selectivity. In the work of Pibiri\net al., 107 individual dye molecules were\nplaced inside a so-called zero-mode waveguide (ZMW) (see Figure 3 d). In this way,\nthe ZMW usage was optimized, and the photophysical properties of dyes\nwere improved compared to stochastically immobilized molecules. DNA objects can also be used as templates or stencils for producing\naccurate objects from different materials. One such example is creating\ncustom-shaped features from inorganic oxides using DNA origami as\na template. 108 In this approach, Surwade\net al. 108 deposited DNA origami structures\non a silicon oxide substrate, and by employing a chemical vapor deposition\nprocedure, they created either oxide layers with DNA-origami-shaped\nopenings or oxide shapes that have inherited the original origami\nshape (positive- and negative-tone patterns). To further employ this\nhigh-resolution and parallel substrate-based technique, Shen et al. 52 showed that these origami-shaped openings in\nthe grown oxide layer can be used as a mask for further lithography\nsteps followed by metal evaporation. They demonstrated the versatility\nof the approach by fabricating gold, silver, and copper nanostructures\nhaving original DNA origami shapes (crosses and rectangles) on the\nsilicon substrate. Later on, Shen et al. 53 generalized the method to other substrates by fabricating, for example,\ngold bow-tie antennas and chiral gold shapes onto silicon nitride\nand sapphire ( Figure 3 e). The authors demonstrated that, by their DNA-assisted lithography\n(DALI) method, it is possible to produce transparent substrates with\nCD properties and a SERS capability for molecular diagnostics. In\nthis work, individual metallic cross-shapes and antennas were also\ncharacterized, and these structures showed the plasmonic resonances\nat the visible wavelength range. However, the main drawback of this\ntechnique is that the metallic structures are randomly oriented on\nthe substrate. Nevertheless, this method is compatible with the approaches\npresented by Gopinath et al., 50 and therefore\nparallel and large-scale patterning of plasmonic substrates with high\nresolution may become possible. DNA origami has also been used\nin optical nanocavity fabrication\nand characterization. Chikkaraddy et al. 109 constructed a nanocavity with <5 nm gap between a Au film and\na AuNP. In such a device, a DNA origami plate between the two components\nhas been employed not only to attach the AuNP to a substrate but also\nto precisely position Cy5 molecules with a nanometer-level lateral\nresolution. This enabled the precise mapping of the local density\nof optical states (LDOS) inside the nanocavity."
} | 7,069 |
34860495 | null | s2 | 5,971 | {
"abstract": "Hydrogels are candidate building blocks in a wide range of biomaterial applications including soft and biohybrid robotics, microfluidics, and tissue engineering. Recent advances in embedded 3D printing have broadened the design space accessible with hydrogel additive manufacturing. Specifically, the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) technique has enabled the fabrication of complex 3D structures using extremely soft hydrogels, e.g., alginate and collagen, by assembling hydrogels within a fugitive support bath. However, the low structural rigidity of FRESH printed hydrogels limits their applications, especially those that require operation in nonaqueous environments. In this study, we demonstrated long-fiber embedded hydrogel 3D printing using a multihead printing platform consisting of a custom-built fiber extruder and an open-source FRESH bioprinter with high embedding fidelity. Using this process, fibers were embedded in 3D printed hydrogel components to achieve significant structural reinforcement (e.g., tensile modulus improved from 56.78 ± 8.76 to 382.55 ± 25.29 kPa and tensile strength improved from 9.44 ± 2.28 to 45.05 ± 5.53 kPa). In addition, we demonstrated the versatility of this technique by using fibers of a wide range of sizes and material types and implementing different 2D and 3D embedding patterns, such as embedding a conical helix using electrochemically aligned collagen fiber via nonplanar printing. Moreover, the technique was implemented using low-cost material and is compatible with open-source software and hardware, which facilitates its adoption and modification for new research applications."
} | 417 |
28275735 | PMC5332151 | pmc | 5,975 | {
"abstract": "By adding a small amount of a vesicle surfactant, “unavoidable” splashing is considerably reduced on superhydrophobic surfaces.",
"conclusion": "CONCLUSION In conclusion, although we have mainly focused on a specific microstructured/nanostructured superhydrophobic surface with varying tilted angles to elucidate the role of the vesicle surfactant (AOT) in inhibiting the receding splash, the scarce or gentle receding behavior can also be generalized to apply to other artificially fabricated superhydrophobic surfaces and other single-drop impact and spray processes at varied impact velocities ( Fig. 4 and movies S6 to S9). In addition, AOT is shown to be a stable surfactant molecule (fig. S6). This work helps advance our understanding of how to control liquid deposition on superhydrophobic surfaces. Therefore, this approach can potentially be used to improve the efficiency of pesticide spraying and to reduce environmental pollution. Fig. 4 The splash inhibition of vesicle AOT generalizes to other superhydrophobic surfaces. ( A 1 and A 2 ) SEM images of silicon nanowire–arrayed surface from top and side views. ( C 1 and C 2 ) SEM images of patterned superhydrophobic surfaces with arrayed silicon micropillars and nanowires from top and cross-sectional views. ( E and G ) SEM images of the microstructured/nanostructured superhydrophobic SiO 2 surface and the superhydrophobic CuO surface with nanoneedles. ( B 1 , D 1 , F 1 , and H 1 ) The impacting water drop makes a big splash on superhydrophobic surfaces. ( B 2 , D 2 , F 2 , and H 2 ) The receding splash is greatly inhibited by 1% AOT on these superhydrophobic surfaces. The impact velocity of each impacting drop is 2.53 ± 0.11 m s −1 .",
"introduction": "INTRODUCTION Pesticide spraying is “hard” agricultural work, where more than 50% of agrochemicals are lost because of undesired bouncing and splashing behaviors on crop leaves with “waterproof” properties ( 1 – 3 ). In natural plants, superhydrophobic leaves are ubiquitous, and they usually get their nonwetting properties from the presence of waxy features on their surface. Particularly because of the combination of extremely low energized chemical composition and microstructured/nanostructured surface morphology, the superhydrophobic surface was demonstrated to facilitate droplets, even those with surfactant additives ( 4 ), bouncing and splashing within shortened contact time ( 5 – 17 ). This increases the difficulty of droplet retention ( 18 ), thus threatening ecological security and human safety: If pesticides cannot be properly deposited on crops, then pests might not be controlled and plant injury might occur ( 1 ). Redundant pesticides might contribute to soil, air, or water pollution, and human health might be negatively affected. Although adding surfactants to the sprayed liquid is considered to be a simple method to reduce surface tension and to improve drop retention on a smooth hydrophobic surface, surfactant liquids still slide or bounce off the hydrophobic surface at a tilted angle and reduce liquid splashing on the superhydrophobic surface ( 19 ). In addition, according to the Kelvin-Helmholtz instability, the wave number k max equals 2ρ a U 2 /3γ, where ρ a is the air density, γ is the fluid surface tension, and U is the relative velocity between gas and liquid. Therefore, reduced surface tension was believed to play a major role in the increased instability of the spilt droplet ( 20 , 21 ). Enhancing liquid deposition on the superhydrophobic surface is complex and difficult work, where selected surfactants need to diffuse from the bulk to the newly created interfaces quickly, because the contact time is typically only several milliseconds, and need to decrease retraction velocity to reduce instability due to the reduced surface tension. Several studies have demonstrated the difficulty of surfactant drop deposition on the superhydrophobic surface. On the basis of these results, it would seem that surfactant additives should not be responsible for reducing high-speed liquid splashing on the superhydrophobic surface. However, we found that this kind of “unavoidable” bouncing or splashing behavior was greatly inhibited or even completely reduced on the superhydrophobic surface at varied angles by adding a small amount of a double-chain vesicle surfactant. Unlike the micelle surfactant previously discussed, the vesicular surfactants additive demonstrated here was able to diffuse from the bulk to the newly formed interfaces during the deformation process, enter into the microstructured/nanostructured morphology, confine the motion of the liquid with the help of the wettability transition during the first inertial spreading stage of ~2 ms, decrease the retraction velocity down to nearly zero, and reduce bouncing and splashing ( Fig. 1 ). For the first time, we have found that the vesicle surfactant could exhibit a distinguished ability to alter the surface wettability during the impact process. By taking advantage of this wettability transition, not only have liquid splashing and retraction been completely reduced but the maximum wetting area is also maintained after the impact process. This behavior is different from that observed in previous research, in which a micelle surfactant drop could bounce off the superhydrophobic surface ( 18 ), viscosity induced the splashing reduction ( 1 ), a surfactant drop partly reduced the liquid retraction on the hydrophobic surface ( 22 – 24 ), and nanoparticles or surface charges suppressed droplet rebound on the superhydrophobic surface ( 19 , 25 ). Fig. 1 The deposition of high-velocity impacting drops on the superhydrophobic leaf surface. ( A ) Optical image of the B. oleracea L. leaf. ( B and C ) Environmental scanning electron microscope (SEM) images of the leaf surface with a microstructured/nanostructured morphology. ( D ) Water contact angle reveals the superhydrophobic property of the leaf surface. ( E ) Impact process of a water droplet on the superhydrophobic leaf surface. The splashing of water mainly occurs in the receding stage. ( F and G ) Inhomogeneous receding behavior: SDS (1%) and TS (1%) additives partially inhibit the receding splash. ( H ) Reduced receding behavior by the AOT surfactant: The receding splash was substantially depressed by 1% AOT additive.",
"discussion": "DISCUSSION Compared with the mechanism of liquid deposition enhancement using polymer additives ( 1 ), surfactant additives cannot alter viscosity but can reduce the liquid’s surface tension. Although surfactants can decrease the surface tension of the liquid, helping it spread on a hydrophobic surface under a low-speed impact ( 25 ), the reduction of surface tension also plays a major role in the increased instability and the enhanced droplet’s splash ( 20 , 21 ). According to the Kelvin-Helmholtz instability, k max = 2ρ a U 2 r /3γ, the key to reducing instability is via the retraction velocity U r , and the brevity of impact contact time should be enough for liquid droplets to wet the superhydrophobic surface ( 18 ). In our experiment, local pinning is observed for SDS ( Fig. 1F ) and TSs ( Fig. 1G ), and the entire pinning is found for AOT in the peripheral area of maximum spreading ( Fig. 1H ), where the retraction velocity U r slows down to a low value, resulting in a small k max . For the AOT drop, the motion of spreading is greatly confined, leading to extremely low instability and thus retarding the splash (movie S1). The exceptional molecular structure of AOT distinguishes it from the other two surfactants in reducing splash and in enhancing liquid deposition. Cryo-TEM (transmission electron microscope) imaging was used to prove our assumption, which was achieved by allowing a free-falling surfactant drop to impact the Cu mesh followed by immersion in liquid nitrogen. This mimics the surfactant packing stage during the impact process. The significant differences of the surfactant aggregates are shown in Fig. 2 (A 1 to C 1 ), where the multilamellar vesicles were closely packed at the air/water interface for the AOT drop with a mass fraction of 1%, whereas micelles only randomly and loosely existed for the other two surfactant solutions at the same mass fractions. Compared with the sample molecular structures of SDS, TSs, and the previously mentioned surfactants in reducing the liquid bouncing ( 18 , 25 ), AOT has two alkyl chains and a relatively small hydrophilic head group. This particular molecular structure of AOT is the main reason for its compact and directed alignment and leads to the multilamellar vesicle structure ( 26 ). As shown in Fig. 2 (A 2 to C 2 ), among the three surfactants, the aqueous solution containing 1% AOT exhibits the lowest DST within a surface age of 80 ms. At the beginning of the bubble pressure measurement of ~10 ms, its DST could decrease to a low value of ~32 mN/m ( Fig. 2C 2 ), whereas both 1% SDS and 1% TSs have DSTs that begin in a high value of ~43 mN/m ( Fig. 2 , A 2 and B 2 ). Similar to the property reflected in the diffusion coefficients achieved through 1 H nuclear magnetic resonance (NMR) spectrometry (fig. S2) and dynamic contact angles (fig. S3), the DST results indicate that AOT has the fastest diffusion speed to the air/water interface and thus has the strongest ability to reduce the surface tension when there are newly created surfaces. Fig. 2 The molecular structure, dynamic surface tension, and impact behavior of SDS, TSs, and AOT. ( A 1 and B 1 ) Cryo-TEM images of both 1% SDS and 1% TSs show the micelle aggregates at a mass fraction of 1%. ( C 1 ) Cryo-TEM image of 1% AOT shows that multilamellar vesicles have formed, indicating the dense aggregates of AOT molecules at the air/water interface. ( A 2 ) SDS has a long chain, and its dynamic surface tension (DST) slowly falls in the rapid fall region and cannot reach its equilibrium within a surface age of 100 ms. ( A 3 ) The droplets containing SDS are easy to rebound on the superhydrophobic surface even for highly concentrated solution. ( B 2 ) TSs have long induction periods in the first tens or hundreds of seconds. ( B 3 ) With the increase of the concentration, the induction period is shortened so that the impact behavior turns from bouncing, emission, to no rebound. On the inclined surface, it is still easy to partially rebound. ( C 2 ) AOT distinguishes itself from other surfactants in the fastest diffusion speed to form a densely packed molecular layer at the air/water interface. AOT directly approaches low surface tension of around 32 mN/m at the very beginning of the bubble pressure measurement, showing the lowest DST among the three surfactants within a surface age of 80 ms. The DSTs of 1% SDS and 1% TSs begin nearly at the same value of ~43 mN/m. TS quickly decreases to a lower value, and SDS slowly decreases to a higher value. ( C 3 ) AOT is the most effective surfactant that inhibits bouncing of the liquid drops on the superhydrophobic surface. Besides reducing splashing on natural superhydrophobic leaves, AOT is the most effective surfactant that inhibits the bouncing and splashing of the liquid drops on the artificial superhydrophobic surface at both low (~1.2 m s −1 ) and high (~2.5 m s −1 ) impact speeds. The artificial superhydrophobic surfaces include a microstructured/nanostructured superhydrophobic surface composed of 20-nm hydrophobic SiO 2 nanoparticle composites with a typical size and spacing of around 200 nm and a CuO nanosheet structured superhydrophobic surface with a typical size of about 3 to 6 μm in length and 200 to 600 nm in width. The water contact angles of these artificial superhydrophobic surfaces are 161.3 ± 0.5° and 159.1 ± 1.7°, respectively, which are much higher than those of the natural B. oleracea L. leaf. A fluorinated glass slide with a water contact angle of 112.8 ± 1.1° is also used for comparison. As shown in movie S5, although the droplets containing SDS can properly deposit on a smooth hydrophobic surface, it is difficult to reduce the rebound on a superhydrophobic surface, no matter how low or how high the impact speed is, even for highly concentrated solutions. For TSs, with the increase in concentration, the induction period is shortened so that the low-speed impact behavior turns to emission from bouncing, whereas it is still easy to partially rebound after a high-speed impact. In contrast, for AOT, the liquid droplets can deposit on the hydrophobic surface at a low concentration of 0.1% and at any superhydrophobic surfaces with a concentration of 0.3% (movie S5). A diagram is shown in Fig. 3 to explain how the receding splash can be substantially depressed by AOT. Driven by the inertia, the impacting liquid first spreads to a maximum diameter ( 27 ). As shown in the spreading state of the diagram, the liquid droplet experiences a large surface deformation during the high-speed impact, and the curved edge of the spreading drop is completely out of equilibrium when it reaches its maximum diameter. Then, surface tension acts on the liquid to retract the flow above the substrate. For water, the drop would break up into multiple droplets during the receding state and would splash in the final state ( Fig. 1E ). For the surfactant drops, if the surfactant molecules cannot replenish the newly created air/water interface in time, typically with high DST, then the surface tension of the deformed drop could not be uniform, where nonuniform receding behavior occurs ( Fig. 3B ). Examples can be found for the SDS drop ( Fig. 1F ), the TS drop ( Fig. 1G ), and the AOT drop in the micelle region (fig. S4). In contrast, if the surfactant with a low beginning DST can effectively saturate the newly created surface within ~1.8 ms (corresponding to the spreading time) and maintain the homogeneous low surface tension at the air/liquid/solid interface, then the liquid can uniformly deposit on the superhydrophobic surface ( Fig. 3C ). As shown in Fig. 1H and fig. S4, a gentle and uniform receding contact line will be obtained similar to AOT in the vesicle region. These results provide direct evidence for the role of AOT in controlling the receding splash. Fig. 3 Schematic illustration for splash inhibition on the superhydrophobic surface by surfactant additives. ( A ) The impacting water drop mainly breaks up in the receding stage after spreading to the maximum lamellar liquid. The upward increased capillary force induced by the squeezed air entrapment in the nanostructure easily makes the water drop take off the surface. ( B ) For the surfactants in the micelle region, the surfactant molecules cannot replenish the newly created air/water interface (high DST), and the surface tension of the deformed drop is not uniform, explaining the nonuniform receding behavior. The nonuniform surface tension leads to partial wettability transition, and several scattered fragments stick on the substrate as SDS, TSs, and AOT in the micelle region. The reduced surface tension makes the entry of the impacting drop in the nanostructure much easier because of the reverse of capillary force. ( C ) Because of the lowest DST and dense aggregates at the air/water interface, AOT in the vesicle region effectively saturates the newly created surface within a short time and maintains a homogeneous low surface tension at the air/liquid/solid interface so that it can change the surface wettability as long as the drop contacts the surface, thus leading to hardly receding behavior and a large wetting area. The underlying mechanism for the abovementioned transient knockdown of the receding velocity at the pinning area can be ascribed to the wettability transition in the spreading phase. At the peripheral area of maximum spreading, the impacting water drop slides over air cushions that are trapped on or beneath the superhydrophobic surface, and it is difficult for the water to enter the nanostructures ( Fig. 3A ). The upward increased capillary force induced by the squeezed air entrapment in the nanostructures easily makes the water drop take off the surface ( 28 ). Similar behavior is observed for the micelle surfactant drops ( Fig. 3B ). The final “floating” state of the micelle surfactant drop, the 0.1% AOT drop, indicates that micelle surfactant drops could not properly reverse surface wettability, which can be seen from the cryo-SEM image in fig. S5. In contrast, the reduction of surface tension induced by the vesicle surfactant leads to the dropdown, reverses the capillary force, and makes an easier and deeper entry of the impacting drop in the nanostructure (the side view in spreading state in Fig. 3C ). Cryo-SEM was used to prove the reverse of surface wettability during the impact, where the vesicle surfactant drop (1% AOT drop) is trapped between the gaps of nanoneedles and fully wets the nanostructured superhydrophobic surface (fig. S5). The outward hydrophobic tails of the surfactants at the air/liquid interface act as bridges to connect the drop and the nanostructure by hydrophobic force and to change the wettability of the superhydrophobic surface. Through this process, the surfactant droplets can be pinned and thus reduce the receding velocity via the wettability transition at the peripheral area of maximum spreading. As a result, the high-speed impacting AOT drops can firmly and quickly deposit on the superhydrophobic surface. The wettability transition at the central contact point is easier than at the peripheral area because capillary forces are overcome by inertial effects ( 29 ) at a high Weber number regime (We > 200). Both the water drop and the surfactant drop tend to become convex in the nanostructure of the superhydrophobic surface because of the downward hammer pressure and the dynamic pressure ( 8 ). However, the water repellency of structure chemistry and the huge upward Laplace pressure induced by the squeezed air entrapment in the nanostructure rebound the water drop, as shown in Fig. 1E . AOT also manifests itself in inhibiting rebound and splash on tilted superhydrophobic surfaces. Superhydrophobic surfaces with tilted angles of 30 o , 60 o , and 75 o are used. In the experiment, the SDS shows little effect on the liquid deposition within the tested concentration region ( Fig. 2A 3 ), although it is a good choice to inhibit rebound on the hydrophobic surface, as shown in movie S5 ( 15 , 16 , 23 ). The TS drop shows the impact behavior from bouncing, emission, to no rebound as the concentration increases from 0.01 to 1%, but it still rebounds partially on the oblique superhydrophobic surface ( Fig. 2B 3 ). The percentage of liquid that bounces off the surface can be quantitatively measured through an analytical microbalance, and the impact processes of surfactant drops on the horizontal and tilted superhydrophobic surfaces are shown in movie S3. Only AOT can suppress the rebound of aqueous droplets on both horizontal and oblique surfaces at a low mass fraction of 0.3% at any inclined angles ( Fig. 2C 3 ). Figure S1 depicts the impact behaviors of aqueous drops containing the AOT additive in three regions. At concentrations lower than the critical micelle concentration, complete bouncing occurs along with complete receding behavior. At the micelle region, partial receding of the contact line is accompanied with partial splashing, partial rebound, and no rebound. Scarce receding of the contact line takes place only at the vesicle region, indicating that both the rebound and receding splashes have been greatly inhibited."
} | 4,890 |
40022248 | PMC11871721 | pmc | 5,976 | {
"abstract": "Background Astaxanthin is a red pigment required by feed, nutraceutical, and cosmetic industries for its pigmentation and antioxidant properties. This carotenoid is one of the main high-value products that can nowadays be derived from microalgae cultivation, raising important industrial interest. However, state-of-the-art astaxanthin production is the cultivation of the green alga Haematococcus pluvialis (or lacustris ), which faces high costs and low production yield. Hence, alternative and efficient sources for astaxanthin need to be developed, and novel biotechnological solutions must be found. The recently discovered cyanobacterium, Synechococcus sp. PCC 11901 is a promising photosynthetic platform for the large-scale production of high-value products, but its potential has yet to be thoroughly tested. Results In this study, the cyanobacterium Synechococcus sp. PCC 11901 was engineered for the first time to our knowledge to produce astaxanthin, a high-value ketocarotenoid, by expressing recombinant β-ketolase (bKT) and a β-hydroxylase enzymes (CtrZ). During photoautotrophic growth, the bKT-CtrZ transformed strain (called BC) accumulated astaxanthin to above 80% of the total carotenoid. Moreover, BC cells grew faster than wild-type (WT) cells in high light and continuous bubbling with CO 2 -enriched air. The engineered strain reached stationary phase after only 4 days of growth in an airlift 80-mL photobioreactor, producing 7 g/L of dry biomass, and accumulated ~ 10 mg/L/day of astaxanthin, which is more than other CO 2 -consuming multi-engineered systems. In addition, BC cells were cultivated in a 330-L photobioreactor to link lab-scale experiments to the industrial scale-up. Conclusions The astaxanthin volumetric productivity achieved, 10 mg/L/day, exceeds that previously reported for Haematococcus pluvialis, the standard microalgal species nowadays used at the industrial level for astaxanthin production, or for other microalgal strains engineered to produce ketocarotenoids. Overall, this work identifies a new route to produce astaxanthin on an industrial scale. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-025-02626-5.",
"conclusion": "Conclusions The metabolic engineering approach herein reported in Syn11901 lead to Asta production at rates of ~ 10 mg/L/day under photoautotrophic growth conditions without the need for stress conditions such as nutrient starvation. Moreover, Syn11901 produces phycocyanin, another industrial-relevant product with different applications in the food and cosmetics sectors. Thus, a biorefinery process to produce ketocarotenoids and phycocyanin in the Syn11901 BC strain is a promising industrial strategy. Furthermore, considering the high photosynthetic efficiency of this fast-growing strain, its cultivation could be integrated with CO 2 -emitting processes for carbon sequestration and conversion into high-value products such as Asta.",
"discussion": "Discussion The advancements in synthetic biology promoted the generation of alternative microalgal strains [ 15 – 21 , 40 , 41 ], which were genetically modified with the aims of synthesizing non-native Asta and, most importantly, overcoming limitations of H. lacustris . Cyanobacterial platforms had the advantage over eukaryotic microalgae of efficiently synthesizing a smaller pool of carotenoids, mainly βcar and Zea, the precursors of microalgal Asta [ 22 ]. The first step of the experimental effort described in this manuscript was inserting of the bKT construct (Fig. 2 a) in the acsA locus, whose sequences for HR were already available [ 2 ]. The obtained transformant was characterized by a brownish pigmentation (Fig. 3 a), similarly to other engineered microalgae accumulating ketocarotenoids [ 15 – 21 , 40 – 42 ]: accordingly, HPLC analysis demonstrated the main accumulation of Cantha in the bKT transformant, while Asta was found only as a minor fraction of total carotenoids. This finding demonstrates that the activity of the endogenous hydroxylation activity of CrtZ enzyme was limiting the conversion of Cantha to Asta. Thus, a second round of transformation was conducted, replacing the kanamycin-resistance cassette of bKT transformant with crtZ from Brevundimonas sp. SD-212 [ 17 ] and smR genes, with the latter conferring resistance to spectinomycin, in an operon configuration. The choice for the use of crtZ from Brevundimonas sp. SD-212 over other possible βcar hydroxylases was due to previous literature about the efficiency of this enzyme in converting Cantha to Asta [ 17 , 33 ]. Moreover, the prokaryotic origin of the crtZ gene herein adopted mitigate the risk of inefficient heterologous gene expression observed in some cases expressing eukaryotic genes in cyanobacteria [ 43 , 44 ]. HPLC analysis confirmed that Cantha was almost absent in BC extract, being entirely replaced by Asta (Fig. 3 g). Remarkable was that the SDS-PAGE analysis of total protein extracts from the evaluated lines (Supplementary Figure S3, Additional File 1 ) showed no bands attributable to the recombinant bKT and CrtZ enzymes. This suggested low accumulation of the heterologous enzymes under the control of Pcpt promoter [ 2 , 26 ]. Nevertheless, transcripts analysis demonstrated that the enzymes were expressed (Supplementary Figure S3c, Additional File 1 ) successfully redirecting carotenoid biosynthesis. Anyway, other genetic tools could be evaluated in Syn11901 to further boost recombinant enzymes expression because the success of synthetic biology approaches generally requires true overexpression of pathway enzymes and proteins of interest to attain higher yields and lower costs [ 44 ]. Importantly, the BC line displayed the fastest growth in exponential phase, despite replacement of acsA (Fig. 4 ). Thus, the presence of astaxanthin did not negatively impact growth of the BC line despite the strong reduction of Zea and βcar in the Syn11901 BC strain. Zea is usually found in lipid membranes having a role in photoprotection in cyanobacteria [ 45 ], but this role is likely complemented by Asta in Syn11901 BC strain. βcar is essential for photosystems assembly, but the residual βcar is likely sufficient for ensuring the accumulation of the photosynthetic complex required for photoautotrophic growth of Syn11901 BC strain. Rather, Syn11901 BC was characterized by a ‘boost’ in the initial stages of growth, with a faster growth rate in the exponential phase compared to WT. This effect is consistent with previous growth data for C. reinhardtii engineered to accumulate Asta [ 46 ] and could be due to: (1) the antioxidant properties of astaxanthin protecting cells and photosystems in the early exponential phase, where the culture is relatively dilute [ 46 , 47 ] and/or (2) the reduced amount of chlorophyll per cell (Table 2 ), conferring a pale-green-like phenotype, which favors greater light penetration, thereby enhancing photosynthetic efficiency [ 34 ]. The yield of Asta produced under the best conditions tested resulted in 38.4 mg/L after 4 days of cultivation (Table 3 ) at an average rate of production of ~ 9.6 mg/L/day, which exceeds that for H. pluvialis , with reported yields in the range of 0.12–4.4 mg/L/day using tubular or bubble columns [ 48 ]. Even if a higher production yield could be obtained for H. pluvialis in more complex cultivation systems [ 49 , 50 ], increasing light availability could further increase the Asta production yield even in the case of the BC strain herein reported. Asta production in Syn11901 is also substantially higher than Asta heterologously synthesized in the cyanobacterium Synechocystis sp. PCC 6803 (2.8 mg/L/day [ 21 ]) and the green alga C. reinhardtii (6.96 mg/L/day; [ 32 ]). Engineered yeasts produce Asta at rates of 37.5 mg/L/day [ 51 ], but growth is heterotrophic and relies on adding glucose to the media. A summary of the production yield obtained in this work compared to other systems is reported in Table 5 . One of the advantage in using Syn11901 compared to other systems is the fact that most carotenoids are present in this species as zeaxanthin and βcar. These molecules are the substrates of the bKT and CtrZ enzymes introduced, allowing for efficient production of Asta (Fig. 1 ). Other carotenoids are strongly accumulated in other systems, such as Chlamydomonas reinhardtii or Nannochloropsis , providing competitive sinks for the precursors needed for astaxanthin biosynthesis. As reported in Table 3 , the lowest Asta content per dry weight was observed in cells grown at the lowest irradiances in 80-ml photobioreactors or in cells grown on a 330-L scale, where light penetration is strongly limited (Table 3 ): exposure to sufficient light is thus needed to boost astaxanthin content per dry weight. Even if nutritional stress did not provide any improvement in Asta titer (Table 4 ), we cannot exclude that other stressing conditions might somehow improve carotenoids biosynthesis. Table 5 Summary of the astaxanthin productivity and content per dry weight in different production systems Genotype Productivity Asta/dcw References (mg/L/day or mg/m 2 /day) % Synechococcus sp. PCC 11901 9.6 mg/L/day; 165.4 mg/m 2 /day 0.59 This work Haematococcus pluvialis up to 4.4 mg/L/day; up to 1.8 [ 48 ] Haematococcus pluvialis 204 mg/m 2 /day 3.8 [ 50 ] Haematococcus pluvialis 15.84 mg/L/day 4.9 [ 49 ] Synechocystis sp. PCC 6803 2.8 mg/L/day 0.30 [ 21 ] Chlamydomonas reinhardtii 6.96 mg/L/day 0.45 [ 32 ] Nannochloropsis oceanica 9.9 mg/L/day in fed-batch mode 0.73 [ 41 ] Saccharomyces cerevisiae 37.5 mg/L/day 1.4 [ 51 ] Yarrowia lipolytica 275 mg/L/day in fed-batch mode 4.1 [ 56 ] There is also scope to improve the production of Asta in the BC strain through additional metabolic engineering and improved PBR design. For instance, levels of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) which are the precursors of carotenoids and other terpenoids could be enhanced [ 21 , 32 , 44 , 52 – 54 ]. Given that deletion of acsA might have a potential negative impact on the growth of the BC strain new loci for insertion of the bKT and crtZ genes could also be tested [ 4 ]. Regarding the cultivation of the engineered strain, the scaling-up costs can be reduced thanks to the recent isolation of a cobalamin-independent strain of Syn11901 that does not require the addition of vitamin B12 [ 3 ]. Possible cultivation in outdoor systems using natural sunlight could also be considered as a possible strategy to further reduce the production costs, even if the cultivation of GMO strains, such as Syn11901 BC is subject to strict regulation. The high salinity of the MAD medium also represents a barrier for contamination by bacteria, weedy algae/cyanobacteria and other organisms, during the cultivation process. However, this barrier can be further strengthened by introducing the PtxD/phosphite-utilizing system [ 55 ] in Syn11901 , which was shown to allow cyanobacterial productions in non-sterile outdoor reactors, reducing costs. In terms of PBR design, improving light penetration, for example by using a tubular PBR, will improve photosynthetic performances and, consequently, biomass and Asta yields."
} | 2,828 |
27151863 | null | s2 | 5,980 | {
"abstract": "Natural recombination combines pieces of preexisting proteins to create new tertiary structures and functions. We describe a computational protocol, called SEWING, which is inspired by this process and builds new proteins from connected or disconnected pieces of existing structures. Helical proteins designed with SEWING contain structural features absent from other de novo designed proteins and, in some cases, remain folded at more than 100°C. High-resolution structures of the designed proteins CA01 and DA05R1 were solved by x-ray crystallography (2.2 angstrom resolution) and nuclear magnetic resonance, respectively, and there was excellent agreement with the design models. This method provides a new strategy to rapidly create large numbers of diverse and designable protein scaffolds."
} | 198 |
24652367 | null | s2 | 5,983 | {
"abstract": "Polymer networks are critically important for numerous applications including soft biomaterials, adhesives, coatings, elastomers, and gel-based materials for energy storage. One long-standing challenge these materials present lies in understanding the role of network defects, such as dangling ends and loops, developed during cross-linking. These defects can negatively impact the physical, mechanical, and transport properties of the gel. Here we report chemically cross-linked poly(ethylene glycol) (PEG) gels formed through a unique cross-linking scheme designed to minimize defects in the network. The highly resilient mechanical properties of these systems (discussed in a previous publication) [J. Cui, M. A. Lackey, A. E. Madkour, E. M. Saffer, D. M. Griffin, S. R. Bhatia, A. J. Crosby and G. N. Tew, Biomacromolecules, 2012, 13, 584-588], suggests that this cross-linking technique yields more homogeneous network structures. Four series of gels were formed based on chains of 35,000 g mol(-1), (35k), 12,000 g mol(-1) (12k) g mol(-1), 8000 g mol(-1) (8k) and 4000 g mol(-1) (4k) PEG. Gels were synthesized at five initial polymer concentrations ranging from 0.077 g mL(-1) to 0.50 g mL(-1). Small-angle neutron scattering (SANS) was utilized to investigate the network structures of gels in both D2O and d-DMF. SANS results show the resulting network structure is dependent on PEG length, transitioning from a more homogeneous network structure at high molecular weight PEG to a two phase structure at the lowest molecular weight PEG. Further investigation of the transport properties inherent to these systems, such as diffusion, will aid to further confirm the network structures."
} | 423 |
36128660 | PMC10092803 | pmc | 5,985 | {
"abstract": "Summary \n The model heterocyst‐forming filamentous cyanobacterium Anabaena sp. PCC 7120 ( Anabaena ) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O 2 ‐sensitive N 2 ‐fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O 2 (a Mehler‐like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative‐specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO 2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler‐like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon‐concentrating mechanisms and CO 2 fixation. Under ambient CO 2 diazotrophic conditions, Flv3A is responsible for moderate O 2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst‐specific uptake hydrogenase, which led to enhanced H 2 photoproduction under both oxic and micro‐oxic conditions. These results reveal a novel regulatory network between the Mehler‐like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.",
"introduction": "Introduction Filamentous heterocyst‐forming cyanobacteria such as Anabaena sp. PCC 7120 (hereafter Anabaena ) represent a unique group of prokaryotes capable of simultaneously performing two conflicting metabolic processes: O 2 ‐producing photosynthesis in vegetative cells, and O 2 ‐sensitive N 2 fixation in heterocysts (lacking active oxygen‐evolving Photosystem II (PSII)). This ability has evolved through cellular differentiation under nitrogen‐limiting conditions when some vegetative (photosynthetic) cells from the filament transform into specialized heterocyst cells that provide a microaerobic environment suitable for nitrogen (N 2 ) fixation. Hydrogen (H 2 ) gas is naturally produced as an obligatory by‐product of the N 2 ‐fixation process carried out by nitrogenase, which is highly sensitive to oxygen (O 2 ). The natural yield of H 2 inside heterocysts is limited. This is due to rapid H 2 recycling, mainly by an uptake hydrogenase enzyme, which returns electrons for the N 2 ‐fixing metabolism (Bothe et al ., 2010 ; Kosourov et al ., 2014 ). In oxygenic photosynthesis, light excites the specific Chl pairs P680 and P700 at the reactions centres of PSII and Photosystem I (PSI), respectively, allowing oxidation of water and subsequent electron transport to NADP + , via PSII, Cytochrome (Cyt) b \n 6 \n f and PSI complexes in the thylakoid membrane, the soluble electron carrier proteins plastocyanin (PC) and ferredoxin (Fd), as well as ferredoxin:NADP + ‐oxidoreductase (FNR). These electron transport reactions are coupled to ATP synthesis via the generation of a trans‐thylakoid proton motive force (pmf). The NADPH and ATP obtained are then used as reducing power for CO 2 fixation and cell metabolism. Environmental fluctuations in light and nutrient supply might result in the over‐reduction of the photosynthetic machinery. Alleviation of excessive reduction by class‐C flavodiiron proteins (hereafter FDP) has been described in all oxygenic photosynthetic organisms, apart from angiosperms, and red and brown algae (Helman et al ., 2003 ; Zhang et al ., 2009 ; Gerotto et al ., 2016 ; Chaux et al ., 2017 ; Ilík et al ., 2017 ; Jokel et al ., 2018 ; Alboresi et al ., 2019 ; Shimakawa et al ., 2021 ). These proteins act as strong electron outlets downstream of PSI by catalysing the photoreduction of O 2 into H 2 O (the Mehler‐like reaction) (Helman et al ., 2003 ; Allahverdiyeva et al ., 2013 , 2015 ; Santana‐Sánchez et al ., 2019 ). Six genes encoding FDPs have been reported in Anabaena (Zhang et al ., 2009 ; Ermakova et al ., 2013 ). Four of these genes ( flv1A , flv3A , flv2 , and flv4 ) are highly similar to their homologs in Synechocystis sp. PCC 6803 (hereafter Synechocystis ), SynFlv1–SynFlv4. Recently, we demonstrated that SynFlv1 and SynFlv3 function in coordination with, but distinctly from, SynFlv2 and SynFlv4 (Santana‐Sánchez et al ., 2019 ). While the SynFlv1/Flv3 hetero‐oligomer is mainly responsible for the initial fast and transient O 2 photoreduction during a sudden increase in light intensity (Santana‐Sánchez et al ., 2019 ), with Fd acting as the electron donor (Nikkanen et al ., 2020 ; Sétif et al ., 2020 ), SynFlv2/Flv4 catalyzes steady O 2 photoreduction under illumination at air‐level CO 2 (LC). Importantly, a single deletion of any SynFDP strongly diminishes O 2 ‐photoreduction, indicating that O 2 photoreduction is mainly catalyzed by the hetero‐oligomeric forms working in an interdependent manner (Santana‐Sánchez et al ., 2019 ; Nikkanen et al ., 2020 ). The two additional Anabaena FDP proteins, AnaFlv1B and AnaFlv3B, are exclusively localized in the heterocysts (Ermakova et al ., 2013 ). The AnaFlv3B protein was shown to mediate photoreduction of O 2 independently of AnaFlv1B, likely as a homo‐oligomer, playing an important role in maintaining micro‐oxic conditions inside heterocysts under illumination, which is crucial for N 2 fixation and H 2 production (Ermakova et al ., 2014 ). However, research into the role of heterocyst‐specific AnaFlv1B and vegetative cell‐specific FDPs in diazotrophic cyanobacteria remains scarce. Here, we address the physiological relevance of the AnaFlv1A and AnaFlv3A isoforms in the bioenergetic processes in vegetative cells and heterocysts of diazotrophic Anabaena . AnaFlv1A and AnaFlv3A are shown to have a crucial role under fluctuating light intensities, regardless of nitrogen or CO 2 availability, suggesting a functional analogy with homologs in Synechocystis . Importantly, our results provide evidence for distinct functional roles of AnaFlv3A and AnaFlv1A. We showed that by cooperating with AnaFlv2 and/or AnaFlv4, AnaFlv3A can function independently of AnaFlv1A in O 2 photoreduction under low CO 2 conditions. AnaFlv3A was also indirectly linked with H 2 metabolism in heterocyst cells. Our work highlights the complex regulatory network between oxygenic photosynthesis, nitrogen fixation and H 2 photoproduction.",
"discussion": "Discussion Heterocyst‐forming cyanobacteria are considered one of the earliest forms of multicellular filaments. Despite extensive characterization of heterocyst differentiation, little is known about the co‐regulation and interdependence of N 2 fixation in heterocysts and oxygenic photosynthesis in vegetative cells. Under challenging environmental conditions, diazotrophic cyanobacteria must find a balance between photochemical reactions and downstream processes that consume electrons in both cell types. In this study, we used ∆ flv1A and ∆ flv3A mutants of Anabaena to examine the physiological significance of the vegetative cell‐specific AnaFlv1A and AnaFlv3A proteins in the bioenergetic processes of diazotrophic cyanobacteria. We have demonstrated that both AnaFlv1A and AnaFlv3A proteins, presumably as hetero‐oligomers, are required for efficient induction of the Mehler‐like reaction and, consequently, for efficient generation of pmf, and likely for activation of CCM during dark‐to‐light transitions, making FDPs crucial for growth when light intensity rapidly fluctuates. Moreover, AnaFlv3A exhibits an important link to H 2 metabolism inside the heterocyst, as inactivation of this protein results in high H 2 photoproduction even under ambient air. In the absence of AnaFlv1A, AnaFlv3A cooperates with AnaFlv2 and/or AnaFlv4 to mediate O 2 \n photoreduction under LC conditions In line with previous results showing a decrease in the expression of both flv1A and flv3A in Anabaena WT upon shifts to diazotrophic conditions (Ermakova et al ., 2013 ), single deletions of AnaFlv1A or AnaFlv3A did not affect the diazotrophic growth of mutants under continuous illumination (Table 1 ). However, both the AnaFlv1A and AnaFlv3A proteins are indispensable during sudden changes in light intensity, similar to their homologous proteins in other species (Fig. S4a ; Allahverdiyeva et al ., 2013 ; Gerotto et al ., 2016 ; Jokel et al ., 2018 ). Here, we have demonstrated that when both AnaFlv1A and AnaFlv3A proteins are expressed in WT filaments, the rate of the Mehler‐like reaction is rapidly increased during dark‐to‐light transitions, likely due to the activity of AnaFlv1A/Flv3A hetero‐oligomers (Fig. 3 ). Accordingly, the absence of either AnaFlv1A or AnaFlv3A impairs O 2 photoreduction (Fig. 3a ), resulting in a strong reduction of the PQ pool upon illumination (Fig. 1a , 1‐qL parameter in Table 1 ), a decrease in PSII yield (Fig. 1a ), and impairment of PSI and Fd oxidation (Fig. 2 ). This phenotype is exaggerated in the mutant lacking AnaFlv3A, which showed a stronger state‐2‐to‐state‐1 transition and a more severely limited ability to oxidize PSI than the mutant lacking AnaFlv1A (Figs 1 , 2 ; Table 1 ). In contrast to the Synechocystis Δ flv1 mutant (Fig. S10 ), AnaFlv3A can promote O 2 photoreduction in Anabaena Δ flv1A (Fig. 3a ), resulting in only a 67% inhibition of steady‐state O 2 photoreduction under LC growth conditions (Fig. 3a ). The near elimination of steady‐state O 2 photoreduction in the Δ flv1A/flv3A double mutant under LC conditions (Fig. 3a ) and in the single mutants under HC conditions (Fig. 3b ) (where AnaFlv2 and AnaFlv4 are strongly downregulated) prompts us to propose functional AnaFlv3A/Flv2‐4 oligomerization, and/or cooperation between a AnaFlv3A/Flv3A homo‐oligomer and AnaFlv2/Flv4 (homo)hetero‐oligomers. Accordingly, the strong impairment of O 2 photoreduction in Δ flv3A might be due to the inability of AnaFlv1A to function as a homo‐oligomer and/or cooperate with AnaFlv2/Flv4. It is worth emphasising that both ∆ flv1A and ∆ flv3A mutants showed similarly enhanced accumulation of flv2 and flv4 transcripts (Fig. 1c ). As the ∆ flv3A mutant showed an elevated flv1A transcript level (Fig. 1c ), the inhibition of O 2 photoreduction in ∆ flv3A cannot be due to downregulation of other FDPs. No contribution of SynFlv3/Flv3 homo‐oligomers in the Mehler‐like reaction was observed in vivo (Mustila et al ., 2016 ), contrary to findings from in vitro studies which suggested that SynFlv3/Flv3 homo‐oligomers function in NAD(P)H‐dependent O 2 reduction (Vicente et al ., 2002 ; Brown et al ., 2019 ). Instead, a possible photoprotective function of SynFlv3/Flv3 homo‐oligomers via an unknown electron transport network has been proposed (Mustila et al ., 2016 ). In Anabaena ∆ flv1A , AnaFlv3A/Flv3A homo‐oligomers may be involved in controlling cation homeostasis, which in turn may affect the reversible association of AnaFlv2/Flv4 hetero‐oligomers with the thylakoid membrane (Zhang et al ., 2012 ) and, consequently, their involvement in O 2 photoreduction. Another possibility is the involvement of AnaFlv3A/Flv3A homo‐oligomers in the reduction of nitric oxide (NO), similar to the FDP‐dependent photoreduction of NO in C. reinhardtii (Burlacot et al ., 2020b ). As NO has a strong inhibitory effect on PSII activity (Solymosi et al ., 2022 ), its efficient reduction by FDPs could serve a photoprotective or regulatory role. Overall, our results suggest a role for AnaFlv3A in promoting steady‐state O 2 photoreduction under diazotrophic LC conditions in an AnaFlv2/Flv4‐dependent manner. Moreover, under LC but not HC conditions, the lack of both AnaFlv1A and AnaFlv3A resulted in a more severe delay in the induction of O 2 evolution during dark‐to‐light transition in comparison to the lack of AnaFlv3A only (Fig. S13d,e ). This suggests that AnaFlv1A may also function independently of AnaFlv3A in an unknown role that facilitates photosynthetic electron transport under LC. Understanding the potential functions of AnaFlv2 and/or AnaFlv4 in these processes and their interactions with AnaFlv1A and AnaFlv3A requires further investigation. All Anabaena FDP mutants studied here exhibited reduced CCM activity, as deduced from slightly lowered CCM coefficients (albeit significantly in the double mutant only) (Fig. 4b,c ), as well as lowered initial peaks in CO 2 uptake rate during dark‐to‐light transitions (Fig. S13b,c ). Moreover, steady‐state CO 2 uptake was severely diminished in ∆ flv3A in comparison to the WT (Fig. 4a ). This may result from impaired energization of CCM in the absence of AnaFlv3A. The pmf generated by FDPs and cyclic electron transport (CET) has been recently shown to be important for inducing and maintaining CCM activity in C. reinhardtii (Burlacot et al ., 2022 ). As both ∆ flv1A and ∆ flv3A exhibited significantly impaired pmf generation upon dark‐to‐light transitions (Fig. 5a ), we hypothesize that the AnaFlv1A/Flv3A hetero‐oligomer is required to rapidly induce the Mehler‐like reaction, which is important for the generation of pmf and induction of CCM activity during dark‐to‐light transitions. The molecular mechanism of the FDP‐dependency of the CCM requires further investigation, however, as the mechanisms of CCM differ between algae and cyanobacteria (Long et al ., 2016 ). Most importantly, cyanobacteria lack the bestrophin‐like HCO 3 \n − transporters whose energization was recently shown to depend on FDPs and CET in C. reinhardtii (Mukherjee et al ., 2019 ; Burlacot et al ., 2022 ). However, operation of the plasma membrane Na + /HCO 3 \n − antiporters SbtA and BicA in cyanobacterial CCMs depend on the export of Na + from the cytosol by the NhaS3 Na + /H + antiporter (Long et al ., 2016 ). Along with the light‐dependent plasma membrane‐localized H + exchanger PxcA (Sonoda et al ., 1998 ), FDPs may be necessary to energize NhaS3‐dependent Na + import by consuming H + in the cytosol (thus increasing pmf). Moreover, the HCO 3 \n − transporter BCT1 consumes ATP to import HCO 3 \n − (Long et al ., 2016 ), suggesting that efficient activation of the ATP synthase by pmf generation is required for CCM induction in cyanobacteria. In both Anabaena ∆ flv1A and ∆ flv3A (Figs 5b , S3b ), as well as in Synechocystis ∆ flv3 (Nikkanen et al ., 2020 ), the activity of the ATP synthase was diminished during dark‐to‐light transitions, which may impair CCM induction. Compelling evidence was recently provided for coordination and functional redundancy between NDH‐1 and Flv1/Flv3, jointly contributing to efficient oxidation of PSI in Synechocystis (Nikkanen et al ., 2020 ) and in P. patens (Storti et al ., 2020a , b ). NDH‐1‐mediated CET in Anabaena could also partially compensate for a lack of AnaFlv1A and AnaFlv3A, as evidenced by observations of a stronger F 0 rise in both mutants (Fig. S6a ). Unlike Synechocystis cells, Anabaena filaments also express orthologs of PTOX (McDonald et al ., 2003 ), which in C. reinhardtii and vascular plants functions as an electron valve from plastoquinol to O 2 , thereby controlling the redox state of the PQ pool (Houille‐Verges et al ., 2011 ; Saroussi et al ., 2019 ). However, as the addition of the PTOX inhibitor nPG did not eliminate the residual O 2 photoreduction in Δ flv1A (Fig. S12a–c ), it is unlikely that AnaPtox is a major contributor to it. Inactivation of AnaFlv3A leads to enhanced H 2 \n yield in heterocysts even under oxic conditions Elevated photoproduction of H 2 in diazotrophic filaments lacking vegetative cell‐specific AnaFlv3A under oxic (Fig. 3 ) and microoxic conditions (Fig. 6 ) demonstrated bioenergetic interdependence between vegetative cells and heterocysts. The heterocyst‐originated production of H 2 in ∆ flv3A was rapidly induced upon exposure to light and occurred concomitantly with O 2 evolution in vegetative cells (Fig. 3 ). Moreover, the rate of H 2 photoproduction in ∆ flv3A responded positively to an increase in CO 2 availability (Fig. 3b ). In the absence of N 2 , the main substrate for nitrogenase, all electrons can be directed to H 2 production (Hoffman et al ., 2014 ; Wilson et al ., 2021 ) allowing a less costly reaction, whereby only 4 mol of ATP are required to produce 1 mol of H 2 . Removal of the N 2 substrate (by replacement with Ar) led to a 10‐fold increase in the H 2 photoproduction rate in ∆ flv3A , demonstrating nitrogenase‐dependent H 2 photoproduction (Fig. 6d ). A recent report suggested that overexpressing heterocyst‐specific Flv3B leads to more stable microoxic conditions inside the heterocysts, notably increasing the H 2 production yield, presumably via the bidirectional hydrogenase Hox (Roumezi et al ., 2020 ). In contrast to the unidirectional production of H 2 by nitrogenase, Hox catalyses the reversible reduction of protons to H 2 (Bothe et al ., 2010 ). We do not consider the contribution of Hox to the photoproduction of H 2 by the ∆ flv3A mutant, as the net production does not fit the bidirectional nature of the enzyme. Observations of the significant downregulation of hoxH transcripts in ∆ flv3A (Fig. S7b ) further support this assumption. Altogether, these results indicate that the increased light‐induced H 2 photoreduction in ∆ flv3A is mediated by nitrogenase activity. Strikingly, the increase in H 2 photoproduction yield in ∆ flv3A was due to significant downregulation of HupL, the large subunit of the uptake hydrogenase (Fig. 6c,f ). The absence of functional Hup suppressed the H 2 recycling pathway (Fig. 6e ) and caused a release of H 2 from heterocysts of ∆ flv3A filaments (Figs 3 , 6 ). Although the Hox hydrogenase can also potentially contribute to H 2 recycling, its input to the process is unclear. Despite hoxH transcripts in all mutants being downregulated (Fig. S7b ), the low level of hydrogen deuteride (HD) formation in samples (Fig. S2 ) indicates the involvement of Hup in H 2 recycling, where the reversible component (H/D exchange) is less pronounced. Our results thus highlight a regulatory network between the two metabolic processes in different compartments: the Flv3A‐mediated metabolic processes in vegetative cells and the H 2 metabolism in heterocysts. It may be that the amount of reducing equivalents in vegetative cells, affected by the activity of Flv3A, has a regulatory role on H 2 metabolism in heterocysts. However, the nature of the molecular signal that would ultimately regulate gene expression in heterocysts remains unknown. While in vivo evidence in Anabaena is lacking, reducing equivalents and metabolites may be interchanged between vegetative cells and heterocysts, inducing changes in metabolism and gene expression (Malatinszky et al ., 2017 ). A majority of the NADPH needed for the nitrogen metabolism in heterocysts derives from the oxidative pentose phosphate pathway breaking down carbohydrates imported from vegetative cells (Cumino et al ., 2007 ), but it is plausible that a more direct exchange of cofactors also occurs, analogously to the malate redox shuttle between the cytosol and chloroplasts in plants and algae. On the other hand, computational modelling suggests that N 2 uptake rate in heterocysts is limited by the supply of fixed carbon (glutamate) from vegetative cells for incorporation of ammonia (Malatinszky et al ., 2017 ). A diminished CO 2 fixation rate (Fig. 4a ) and, possibly, a decrease in GOGAT activity in vegetative ∆ flv3A cells may therefore induce changes in heterocyst H 2 metabolism and gene expression. Nevertheless, the molecular mechanism underlying the regulatory network between different cell types needs further elucidation. Taken together, our results demonstrate that vegetative‐cell‐specific AnaFlv1A and AnaFlv3A are indispensable under harsh fluctuating light conditions regardless of nitrogen or CO 2 availability, most likely maintaining sufficient oxidation of the photosynthetic electron transport chain and pmf generation for ATP synthesis, and allowing energization of the CCM by catalysing the Mehler‐like reaction as AnaFlv1A/Flv3A hetero‐oligomers. Under LC, AnaFlv3A facilitates moderate O 2 photoreduction independently of AnaFlv1A in coordination with AnaFlv2 and AnaFlv4. Deletion of AnaFlv3A caused downregulation of the heterocyst‐specific Hup enzyme, resulting in increased light‐induced net H 2 production. This novel regulatory network between photosynthesis and diazotrophic metabolism might represent an unexploited source of future biotechnological applications."
} | 5,216 |
23355305 | PMC3590773 | pmc | 5,987 | {
"abstract": "Many insect species have established long-term symbiotic relationships with intracellular bacteria. Symbiosis with bacteria has provided insects with novel ecological capabilities, which have allowed them colonize previously unexplored niches. Despite its importance to the understanding of the emergence of biological complexity, the evolution of symbiotic relationships remains hitherto a mystery in evolutionary biology. In this study, we contribute to the investigation of the evolutionary leaps enabled by mutualistic symbioses by sequencing the genome of Blattabacterium cuenoti , primary endosymbiont of the omnivorous cockroach Blatta orientalis, and one of the most ancient symbiotic associations. We perform comparative analyses between the Blattabacterium cuenoti genome and that of previously sequenced endosymbionts, namely those from the omnivorous hosts the Blattella germanica (Blattelidae) and Periplaneta americana (Blattidae), and the endosymbionts harbored by two wood-feeding hosts, the subsocial cockroach Cryptocercus punctulatus (Cryptocercidae) and the termite Mastotermes darwiniensis (Termitidae). Our study shows a remarkable evolutionary stasis of this symbiotic system throughout the evolutionary history of cockroaches and the deepest branching termite M. darwiniensis , in terms of not only chromosome architecture but also gene content, as revealed by the striking conservation of the Blattabacterium core genome. Importantly, the architecture of central metabolic network inferred from the endosymbiont genomes was established very early in Blattabacterium evolutionary history and could be an outcome of the essential role played by this endosymbiont in the host’s nitrogen economy.",
"introduction": "Introduction Symbiotic associations between eukaryotes and prokaryotes are common in nature and have been described in all branches of the eukaryotic tree of life ( Moya et al. 2008 ). Insects characterized by feeding upon unbalanced diets have established symbiotic associations with bacteria that provide nutrients lacking in the diet. This is the case of essential amino acid provision by Buchnera aphidicola , the primary endosymbiont of pea aphids feeding on phloem sap ( Baumann 2005 ; Shigenobu and Wilson 2011 ), whereas the same metabolic function is performed by a consortium comprising the coprimaries Buchnera aphidicola and Serratia symbiotica in the case of the cedar aphid ( Pérez-Brocal et al. 2006 ; Lamelas et al. 2011 ) . However, in insects such as cockroaches, which feed on complex diets, the role of the obligatory endosymbiont, Blattabacterium cuenoti , was unclear before the genomes of Blattella germanica (BBge) ( López-Sánchez et al. 2009 ) and Periplaneta americana (BPam) ( Sabree et al. 2009 ) strains were sequenced. The symbiosis between Blattabacterium and cockroaches may have become established between 300 Ma, the age of the first fossils of roaches in the Carboniferous, and 140 Ma, that is, before the diversification of extant cockroach families in the Cretaceous ( Clark et al. 2001 ; Vrsansky et al. 2002 ; Lo et al. 2003 ). Phylogenetic analyses based on 16S ribosomal DNA have located Blattabacterium as members of the class Flavobacteria in the phylum Bacteroidetes ( López-Sánchez et al. 2008 ). The presence of these endosymbiotic bacteria has been observed in all studied species, with the only known exception being that of cockroaches from the genus Nocticola ( Lo et al. 2007 ). Moreover, in termites, a sister clade of cockroaches, only Mastotermes darwiniensis has retained a symbiont ( Bandi et al. 1995 ). Recently, another two genomes of Blattabacterium strains have been sequenced from wood-feeding hosts, the subsocial roach Cryptocercus punctulatus (BCpu) ( Neef et al. 2011 ), and the termite M. darwiniensis (BMda) ( Sabree et al. 2012 ). All these four Blattabacterium genomes show typical features of other insect primary endosymbionts, such as low GC content, total absence of repeated elements, and a reduced genome size compared to their closest free-living relatives ( McCutcheon and Moran 2012 ). Flux balance analyses (FBA) of the reconstructed metabolic networks from BBge and BPam strains have highlighted the functional equivalence of both networks, despite a prolonged divergence time and a few remarkable enzymatic differences, such as the absence of the first three steps of the tricarboxylic acid cycle (TCA) cycle in BPam ( González-Domenech et al. 2012 ). Analyses would also suggest the endosymbionts play a role in the host’s nitrogen metabolism ( Feldhaar et al. 2007 ) leading researchers to postulate a mechanism for the intriguing ammonotelism described in cockroaches ( Mullins and Cochran 1972 , 1976 ; Cochran 1985 ). In addition, metabolic inferences of Blattabacterium strains from B. germanica (BBge) and P. americana (BPam) suggest they are involved in supplying essential amino acids and cofactors to the host ( López-Sánchez et al. 2009 ; Sabree et al. 2009 ). Moreover, Blattabacterium strains from all four studied species encode urease genes, and endosymbiont-enriched extracts from P. americana and B. germanica show urease activity ( López-Sánchez et al. 2009 ), thus this activity may be essential for nitrogen recycling from urate deposits ( González-Domenech et al. 2012 ). On the other hand, BCpu and BMda strains retain the enzymes required for nitrogen metabolism, even though they have lost pathways for the synthesis of several amino acids ( Neef et al. 2011 ; Sabree et al. 2012 ). In this work, we report on the genome of the Blattabacterium from Blatta orientalis (BBor), a sister clade of P. americana from the family Blattidae ( Kambhampati 1995 ; Inward et al. 2007 ). Comparative genome analysis of all five genomes has shed light on the evolution of the primary endosymbiont because it became associated with a common ancestor of cockroaches and termites. Of particular interest is the extraordinary conservation of both genome architecture and gene content.",
"discussion": "Discussion The genome sequence of BBor has confirmed the extreme stability of the Blattabacterium genome architecture despite the fact extant cockroach families appeared more than 140 Ma ( Sabree et al. 2010 , 2012 ; Neef et al. 2011 ). The only rearrangements are a 20 kb inversion, which occurred in the Blattidae family lineage, and two inversions in the strain BMda, one of 242 kb and another of 2.9 kb, containing the genes rffH , dut , and wzxC ( fig. 1 ). As in other endosymbiotic bacteria, synteny is highly conserved among the different strains; however, surprisingly, Blattabacterium has also maintained a very similar gene content, given 83.0% of the pan-genome genes are represented in the core ( table 3 ). This conservation is even more striking when only the strains from omnivorous cockroaches are considered: 93.9% of the pan-genome genes are included in the core. Another example of endosymbiotic bacteria of omnivorous hosts is Blochmannia sp., primary endosymbiont of Camponotus sp. ants. In this case also, the number of genes is well conserved because 93.5% of the pan-genome genes are in the core ( Williams and Wernegreen 2010 ). However, the divergence time among the three Blochmannia strains has been established at approximately 20 Ma ( Degnan et al. 2004 ; Gómez-Valero et al. 2008 ), whereas the symbiosis between cockroaches and Blattabacterium originated at least 140 Ma ( Clark et al. 2001 ; Lo et al. 2003 ). Comparison with a similarly ancient symbiotic association, similar to the one established between Buchnera and aphids between 86 and 164 Ma ( von Dohlen and Moran 2000 ), and considering only true primary endosymbionts, that is, those without any known metabolic complementation with other symbionts (as is the case of the cedar and tuja aphids), the genetic conservation is much lower, because several gene losses have occurred in the different lineages. In particular, only 74% of the pan-genome genes are present in the core ( table 3 ). All these data suggest that massive gene losses may have occurred in Blattabacterium genomes soon after the transition from the free-living state to the intracellular life style, establishing an optimal genome to fulfill the host requirements, albeit minimized. We postulate that this gene content stasis may be correlated with the role played by Blattabacterium in the nitrogen metabolism in cockroaches ( González-Domenech et al. 2012 ). In fact, gene losses observed in the Blattidae lineage only affect metabolism in peripheral activities (i.e., the change from sulfate to sulfide as sulfur source) or irrelevant metabolic steps in the Krebs cycle. Hence, one of the most remarkable losses in BBor is the absence of genes coding for the three first steps of the TCA cycle, citrate synthase ( gltA ), aconitate hydratase ( acnA ), and isocitrate dehydrogenase ( icd ). Nevertheless, this metabolic feature shared with BPam ( Sabree et al 2009 ) has no effect on metabolic network functionality as shown by FBA ( González-Domenech et al. 2012 ).\n Table 3 Number of Genes in the Pan-Genome and the Core in Blattabacterium , Blochmannia sp., and Buchnera Genes in Pan-Genome Genes in Core (%) a Blattabacterium Strains: BBge, BPam, BBor, BCpu, and BMda 652 541 (83.0) Onmivorous strains: BBge, BPam, and BBor 644 605 (93.9) Blochmannia Strains: Bfl, Bpen, and Bva 660 617 (93.5) Buchnera Strains: BAp, BKo, BUa, BSg, and BBp 650 481 (74.0) N ote .—Strain abbreviations are as follows. Blochmannia strains: Bfl, Blochmannia floridanus ; Bpen, B. pennsylvanicus ; Bva, B. vafer . Buchnera strains: BAp, Buchnera from Acyrtosiphon pisum ; BKo, Buchnera from A. kondoi ; BUa, Buchnera from Uroleucon ambrosiae ; BSg, Buchnera from Schizaphis graminum ; BBp; Buchnera from Baizongia pistaciae . a Proportion of the pan-genome present in the core genome. Most of the core genes in the Blattabacterium pan-genome (62.1%) follow a molecular clock, which allowed us to determine the split times between the Blattidae lineages and the Termitidae and Cryptocercidae lineages ( fig. 4 and supplementary fig. S1 , Supplementary Material online). The divergence time calculated for the wood-feeding lineages is closer to the split between B. germanica and the rest of the cockroach species (88.6 vs. 140 Ma). This indicates that the metabolic changes taking place in the ancestor of the endosymbionts of M. darwiniensis and C. punctulatus, mainly affecting the biosynthesis of essential and nonessential amino acids ( fig. 4 ), took place very early in the evolutionary history of the lineage leading to Termitidiae and Cryptocercidae. Conversely, B latta orientalis and P. americana are much more recent lineages (split occurring 12.9 Ma) and, as previously stated, the metabolic profile of their common ancestor indicates notable metabolic stasis. Metabolic comparison among Blattabacterium strains highlights the role of the bacterial metabolic network in host physiology. For instance, in the case of nitrogen metabolism, urease corresponds to the Blattabacterium core genome, suggesting that the role of the endosymbiont in urate mobilization is ancestral and conserved in all studied Blattabacterium lineages. Hence, the combination of urease with the urea cycle (interrupted only in BCpu and BMda by the absence of argininosuccinate lyase [ASL]) is a metabolic network with the potential to catabolize nitrogen compounds and generate ammonia ( López-Sánchez et al. 2009 ; González-Domenech et al. 2012 ). Thus, there is a biochemical explanation for the classical model of urate deposits acting as nitrogen storage in the cockroach fat body ( Mullins and Cochran 1974 ; Cochran et al. 1979 ; Cochran 1985 ) and the intriguing ammonotelism displayed by these insects ( González-Domenech et al. 2012 ). With respect to gene losses in the two wood-feeding hosts, a higher number of events have occurred although very early in their evolutionary history. Remarkably, these affected the biosynthesis of essential and nonessential amino acids before the split between Cryptocercidae and Termitidae, and the convergent loss of sulfate assimilation ability in the BCpu lineage. As stated earlier, the symbionts from the three omnivorous cockroaches are self-sufficient in terms of supplying their host with essential amino acids, whereas up to six pathways for essential amino acids have been lost in the wood-feeding hosts. On the basis of the phylogenetic analysis, we propose that a major reduction in amino acid biosynthetic ability took place in the common ancestor of the two wood-feeding lineages. It has been postulated that this metabolic impairment of the endosymbiont could have been compensated for by amino acid supply from the diet and/or their syntheses by gut microbiota ( Neef et al. 2011 ; Sabree et al. 2012 ), comparable with the metabolic complementation observed in some bacterial consortia, like Buchnera and Serratia in cedar aphids ( Lamelas et al. 2011 ). Additionally, the gene argH coding for ASL, the last step in the Arg biosynthesis pathway, has been lost in both BCpu and BMda strains, thus a host-encoded ASL may also have taken over Arg biosynthesis; nonetheless, the contribution of diet or gut microbiota cannot be ruled out. In fact, 8 of the 10 complete genomes from insects accessible in the KEGG database ( http://www.genome.jp/kegg/ , last accessed February 4, 2013) contain genes for ASL. The corresponding gene is absent from the pea aphid Acyrthosiphon pisum genome (but present in its primary endosymbiont Buchnera aphidicola ) nor is it present in the human body louse Pediculus humanus genome (in this case, Arg may be supplied by the blood ingested by the louse). Studies of metabolic modeling supported by transcriptomic and proteomic data ( Macdonald et al. 2012 ) have revealed the action of host cell enzymes at the end of amino acid biosynthesis in the pea aphid and Buchnera aphidicola , which could have important regulatory consequences. There are some remarkable convergent metabolic traits, in particular, the capacity for assimilating sulfate. The symbionts BBge and BMda possess the genes coding for all enzymes involved in sulfate assimilation, with the exception of 5′-phosphosulfate kinase (encoded by cysC ). Experimental data indicate that this pathway may be operative, because aposymbiotic individuals of B. germanica are unable to incorporate sulfate into cysteine and methionine ( Block and Henry 1962 ). As this pathway is absent in the other Blattabacterium strains, it was most likely lost in at least two independent events, one in the lineage leading to BCpu and the other during the evolution of the endosymbionts of Blattidae species ( fig. 5 ). The genes cysN , cysD , and cysI are found as pseudogenes in BPam ( Sabree et al. 2009 ), whereas in BBor, they have been completely lost. With respect to the remaining genes of this pathway, cysH and cysG are pseudogenized in BBor but seem to be functional in BPam. However, gene cysJ is present in all sequenced Blattabacterium strains, even in those which are unable to assimilate sulfate. The product of this gene is a flavoprotein, which forms sulfite reductase with the hemoprotein coded by cysI . In this case, cysJ may have been recruited by other processes, because it has previously been described to work as FMN reductase ( Covès et al. 1993 ). The genomes of BBor, BCpu, and BMda retain seven of the nine duplicated genes found in BBge ( López-Sánchez et al. 2009 ; Neef et al. 2011 ; Sabree et al. 2012 ), whereas eight of these genes are also maintained in BPam ( Sabree et al. 2009 ). The presence of these duplicated genes is quite surprising in the context of a reduced genome, similar to that of Blattabacterium and other primary endosymbionts. The fact most of these genes are still present in all three strains points to their possible functional role in bacterium physiology and that these genes might be ecoparalogs ( Sanchez-Perez et al. 2008 ). In summary, the hosts harboring Blattabacterium strains have been evolving separately for a very long time; however, the genomic and metabolic architecture of these symbiotic bacteria remains strikingly stable. Thus, the basic genetic and metabolic traits of Blattabacterium were established in a short time period, when these bacteria infected the common ancestor of all cockroaches and termites."
} | 4,137 |
26598941 | null | s2 | 5,989 | {
"abstract": "Microorganisms are the pillars of life on Earth. Over billions of years, they have evolved into every conceivable niche on the planet. Microbes reshaped the oceans and atmosphere and gave rise to conditions conducive to multicellular organisms. Only in the past decade have we started to peer deeply into the microbial cosmos, and what we have found is amazing. Microbial ecosystems behave, in many ways, like large-scale ecosystems, although there are important exceptions. We review recent advances in our understanding of how microbial diversity is distributed across environments, how microbes influence the ecosystems in which they live, and how these nano-machines might be harnessed to advance our understanding of the natural world."
} | 185 |
33050056 | PMC7601019 | pmc | 5,990 | {
"abstract": "Mechanical property is one of the most important properties of nanofiber membranes. Electrospinning is widely used in the preparation of nanofibers due to its advantages such as good stability and easy operation. Compared with some nature silk, the mechanical properties of nanofibers prepared by electrospinning are poor. Based on the principle of vortex spinning and DNA structure, this paper designed an air vortex electrospinning device that can control the structure of macromolecular chains in nanofibers. When a weak air vortex is generated in the electrospinning process, the macromolecule chains will entangle with each other and form a DNA-like structure so as to improve the mechanical property. In addition, when a strong air vortex is generated during the electrospinning process, the nanofibers will adhere to each other, thereby enhancing the mechanical property and enlarging the pore size.",
"conclusion": "4. Conclusions In this paper, for the first time, we use air vortex in the electrospinning process. Compared with traditional electrospinning and cross-certified through the scanning electron microscopy, mechanical property test, Fourier transform infrared spectroscopy and pore size test, we found that weak air vortex in the electrospinning process could control the macromolecule chains’ structure in a nanofiber, making the macromolecule chains entangled with each other and forming a DNA-like structure. As the intensity of the vortex increases, the diameter of the nanofibers gradually decreases. When the vortex strength increases to a certain value, the entanglement of the macromolecular chains inside the nanofiber reaches the limit, and at the same time, the nanofibers begin adhering to each other. As the vortex intensity increases, the mechanical properties of the nanofiber membrane will also increase. Besides, it is also observed that strong air vortex could not only improve the mechanical property of the nanofiber membrane, but also enlarge the pore size, which provide a new and simple method for fabricating high strength and large pore size nanofiber membrane. In a word, this paper proposes a promising method to control the macromolecule chains structure in a nanofiber and provides an effective and simple method to prepare nanofiber membranes with high strength and large pore size.",
"introduction": "1. Introduction Vortex spinning is a pneumatic spinning method and widely used in the textile industry [ 1 , 2 , 3 ]. It can be viewed as a refinement of jet spinning, or a natural development in fascinated yarn technology, which enables the yarns to exhibit better evenness and tenacity values [ 4 , 5 ]. As we know, DNA has a twisted double helix structure with replication property [ 6 , 7 ]. Similarly, in the yarn spinning, twisting is also one of the most important steps, which influences the geometry of spinning triangle and effects the properties of spun yarns directly. Regarded as the most straight and simplest method to fabricate nanofibers [ 8 , 9 , 10 ], the electrospinning is used to prepare various materials for different applications [ 11 , 12 , 13 ]. On the other hand, as a key factor, the mechanical property will determine the application range of nanofiber membranes [ 14 , 15 , 16 ]. Therefore, how to further improve the mechanical property of nanofiber membrane has always been an important problem for researchers. We tried to control the macromolecule orientation in a nanofiber before, but its mechanical property was very poor [ 17 ]. In this paper, inspired by DNA structure and based on vortex spinning, we used the air vortex to control macromolecule chains structure in a nanofiber, and designed an air vortex electrospinning device for fabricating nanofibers whose macromolecule chains have a DNA-like structure. We also studied and discussed the effect of the air vortex strength on macromolecule chains. This paper aimed at demonstrating that the air vortex has an important effect on macromolecule chains in a nanofiber and between nanofibers in the electrospinning process. So as to improve the mechanical property of nanofiber membranes.",
"discussion": "3. Results and Discussion 3.1. Air Vortex Electrospinning In this part, we discussed the influence of air vortex on nanofibers. PVA and PAN nanofibers were prepared using the air vortex electrospinning device. The electrospinng condition were all the same. The voltage was 18 kV, the flow ratio was 1 mL/h, the distance between the needle and the collector was 20 cm, the temperature was 24 °C, and the relative humidity was 37%. 3.1.1. Morphological Characterization (SEM) Figure 2 shows the SEM images of nanofibers prepared using different electrospinning devices and different polymer solutions. Table 1 shows the average diameter of these nanofibers. It can be seen from Figure 2 that the PVA nanofibers and PAN nanofibers prepared by different electrospinning devices have no significant changes in morphology. This reflects that the air vortex only has an effect on the internal structure of the nanofibers. Through the measurement of the average diameter, we known that the nanofibers prepared by the air vortex electrospinning device have a finer diameter. This is because under the action of the air vortex, the vortex exerts a weak stretching force on the jet during spinning. In addition, since the macromolecular chains in the nanofibers are entangled with each other, their position distribution in nanofibers are more concentrated. Therefore, the space they occupied will be relatively small, and the diameter of the nanofiber will be smaller. Besides, the internal macromolecular chains of a certain concentration of spinnable polymer solution are entangled with each other [ 22 , 23 , 24 ]. So, the high shear stress and elongation stress caused by the vortex flow may reduce the viscosity of the spinning solution, thereby reducing the diameter of the prepared nanofibers. 3.1.2. Mechanical Property The entanglement of macromolecular chains inside nanofibers will greatly affect the mechanical properties of nanofiber membranes. Figure 3 shows the mechanical property test curves of PVA and PAN nanofiber membranes prepared by different electrospinning devices. It can be seen that for the two different polymers, the breaking strength and breaking elongation of the nanofiber membranes prepared by the air vortex electrospinning device are larger than those prepared by the traditional electrospinning device. This is because when the air vortex act on the spinning jet, the macromolecular chains in the jet are entangled with each other, and the macromolecular chains in the prepared nanofibers will also be entangled. So, when the tensile force acts on the nanofiber membranes, the friction force that needs to be overcome due to the entanglement of the macromolecular chains inside the nanofiber will greatly increase, resulting in an increase in the maximum breaking strength and the toughness of the nanofiber membranes. From the SEM image, we can see that the diameter of the nanofibers prepared by the air vortex electrospinning device has some reduced, but there is no adhesion between the nanofibers, the morphology of the nanofibers has little change. In this case, the mechanical properties of the nanofiber membranes have been improved. It is further proved that it is the results of the entanglement of macromolecular chains inside the nanofibers. 3.1.3. Fourier Transform Infrared (FTIR) Spectroscopy Figure 4 present the FTIR spectra of PVA and PAN nanofibers. The infrared spectrum of PVA, the bands at 820 cm −1 attribute to C-C vibration, 1100 cm −1 relate to C-O stretching vibration, C-OH vibration at 1410 cm −1 , 1710 cm −1 attribute to C=O stretching vibration, CH 2 bending and stretching vibration at 2922 cm −1 [ 25 , 26 ]. The infrared spectrum of PAN, the adsorption peaks of stretching vibrations at 2242 cm −1 (C ≡ N), 1732 cm −1 (C=O of ester group), 1446 cm −1 (C-H bending in CH 2 ) and 1245 cm −1 (C-N bending) are clearly observed [ 27 , 28 , 29 ]. From the spectra, since no new band as well as any shift at absorbance bands is observed in nanofibers, it can be concluded that no chemical bonds have been created. However, in the infrared spectrum of nanofiber membranes fabricated by air vortex electrospinning device, some peaks were strengthened, this may be due to the entanglement of macromolecule chains, which enhancing the vibration. 3.1.4. Gas Permeability The pore size distributions of PVA and PAN nonofiber membranes were measured by a capillary flow porometry. Figure 5 and Table 2 illustrate the pore size distributions of these nonofiber membranes prepared by traditional electrospinning device and air vortex electrospinning device. It can be seen from Figure 5 and Table 2 that the pore size of the nanofiber membrane prepared by the air vortex electrospinning device is smaller. This is because the addition of air vortex makes the distribution of nanofibers denser. At the same time, the entanglement of the macromolecular chains inside the nanofibers makes the nanofibers thinner, and the pore size of the nanofiber membrane is positively correlated with the diameter of the nanofibers [ 30 , 31 , 32 ]. When the nanofiber diameter is smaller, the pore size of the nanofiber membrane is correspondingly smaller. However, because the diameter does not change much, the change of the pore size is also small. 3.2. Different Air Vortex Strength In the previous section, we used the air vortex electrospinning device and selected two different polymer solutions for spinning and discussed the influence of the air vortex on the macromolecular chains inside the nanofibers. The previous experiments found that the weak air vortex has a greater impact on the macromolecular chains inside the nanofibers but has almost no effect on the nanofibers. In addition, the use of the air vortex improves the mechanical properties of the nanofiber membranes. In this section, we changed the intensity of the air vortex by selecting different airflow speeds and discussed the influence of air vortex strengths on the macromolecular chains inside the nanofibers and the interaction between nanofibers. In this section, 8% PVA solution was used for spinning, and the speed of the pumped airflow was selected as 1 m/s, 2 m/s, 3 m/s, 4 m/s and 4.5 m/s, respectively. The silk conditions and spinning environment were also consistent with those described above. 3.2.1. Morphological Characterization The change of air vortex strength would have a great impact on the morphology of nanofibers. Figure 6 and Table 3 show the SEM images and average diameters of nanofibers prepared via different air vortex strengths. When the velocity of the air vortex is 0 m/s, it is the traditional electrospinning device. It can be seen from Table 3 and Figure 6 that as the velocity of the pumped airflow increases, the average diameter of the nanofibers gradually decreases. This is because when the speed of the pumped airflow increases, the strength of the air vortex formed in the tube will also increase. We know that the vortex has a certain stretching effect on the electrospinning jet. Therefore, when the strength of the air vortex increases, the stretching force of the air vortex on the jet will also increase, and the diameter of the nanofibers will decrease with the increase of the strength of the air vortex. On the other hand, it can be seen from the SEM images that when the incident air velocity is small, that is, when the air vortex intensity is small, the air vortex has little effect on the interaction between the nanofibers. However, when the speed increased to 3 m/s, the air vortex caused the nanofibers to adhere to each other. When the velocity of the pumped air exceeds 3 m/s, as the incident air velocity increases, that is, as the vortex intensity increases, the adhesion between nanofibers becomes more and more serious. This is because when the strength of the air vortex is small, the air vortex only has an effect on the macromolecular chains inside the nanofibers, but when the strength of the air vortex increases to a critical value, it starts to have an effect on the nanofibers, making the nanofibers adhere to each other. And as the strength of the air vortex increases, this force on the nanofibers will also increase. As shown in Figure 6 f, when the incident air velocity reaches 4.5 m/s, the adhesion between nanofibers has become very serious. From Table 3 , it can be seen that as the intensity of the air vortex increases, the confidence interval was gradually decreasing, which shows that the distribution of nanofibers becomes more and more uniform. 3.2.2. Mechanical Property Test From the SEM images we know that when the air vortex strength is weak, it just has effect on macromolecule chains, but once it increases to a critical value, the air vortex begins have effect between nanofibers. So, the air vortex strength will greatly affect nanofiber membranes’ mechanical property. Figure 7 show the mechanical property of nanofiber membranes prepared in different air vortex strength. It can be seen from Figure 7 a that in general the mechanical property of nanofiber membrane increases with the increase of the air vortex strength. This is because when the air vortex strength increased, the entanglement of macromolecule chains become serious, and at the same time nanofibers’ adhesion become serious, too. So, based on the two reasons, the force to overcome the friction among macromolecule chains and nanofibers adhesion will increase, and the mechanical property will be enhanced. Figure 7 b is the maximum stress of nanofiber membranes prepared in different air vortex strength. From this figure we can see that when the velocity of the airflow increased from 0 m/s to 2 m/s, the stress keeps growing trend, but from 2 m/s to 4 m/s, the stress become steady. This maybe because when velocity increased to 3 m/s, the entanglement of macromolecule chains reaches the limitation, and even though the nanofibers begin adhering with each other, but the adhesion was not very serious. Therefore, the stress has little change. However, when the velocity increased to 4.5 m/s, the adhesion become very serious, we can also see this phenomenon from the SEM. So, the force to overcome the friction among nanofibers increased, and the stress increased rapidly. 3.2.3. Gas Permeability The pore size distributions of PVA membranes obtained in different air vortex strength were measured by a capillary flow porometry. Figure 8 and Table 4 illustrate the pore size distributions of PVA membranes prepared in different air vortex strength. From Table 4 and Figure 8 we can see that when the air vortex is weak (from 0 m/s to 2 m/s), the pore size decreased first when the air vortex strength increased. This is because the vortex mainly acts on the macromolecular chains inside the nanofibers at this time, and the entanglement of the macromolecular chains becomes more and more compact. The nanofibers are also densely distributed under the action of the vortex, so the pore size of nanofiber membrane gradually decreases. However, when the velocity of the airflow increased to 3 m/s, the pore size begins to gradually increase. This is because the vortex has an effect on the nanofibers and makes the nanofibers adhere to each other. Moreover, as the intensity of the vortex increases, the adhesion between the nanofibers becomes stronger and stronger, so the pore size of the nanofiber membrane will increase. In addition, we can see that the average pore size of nanofiber membrane prepared in the velocity of 4 m/s is similar to the nanofiber membrane prepared in the velocity of 4.5 m/s. This is because the adhesion between nanofibers not happen in every nanofiber, so the adhesion between nanofibers will improve the mechanical property of nanofiber membrane but will not enlarge the average pore size."
} | 3,983 |
37763247 | PMC10533193 | pmc | 5,991 | {
"abstract": "Despite the well-documented role of biochar in promoting soil quality and crop productivity, the underlying biological mechanisms remain poorly understood. Here, we explored the effects of straw biochar on soil microbiome in the rhizosphere from wheat using metagenomic sequencing. Our results showed that straw return decreased the yields of wheat, while the straw biochar return increased the wheat yields. Further, both the richness and community composition confirmed different effects of the straw return and straw biochar return. The straw biochar return also resulted in greater rhizosphere effects from wheat, represented by resource availability, including soil organic carbon, soil total nitrogen, available phosphorus, and available potassium. The rhizosphere effects from wheat, represented by microbial metabolism genes involved in carbon, nitrogen, phosphorus, and potassium cycling, however, were decreased by straw biochar returning. In addition, the rhizosphere effects from nitrogen content and the nitrogen cycling genes showed negative relationships with wheat yields. Together, these results revealed that straw biochar enhanced soil resource availability but suppressed microbial metabolism genes in the rhizosphere from wheat, supporting the idea that straw biochar serves as a nutrient pool for crops.",
"conclusion": "5. Conclusions In conclusion, using PCR-bias-free metagenomics sequencing, we found that straw biochar amendments enhanced the rhizosphere effects from wheat on soil available resources, although it suppressed the abundance of microbial metabolism genes. These microbiome variations suggest that biochar functions as a direct nutrient source rather than an indirect method of biological soil engineering in a wheat-growing agroecosystem. Thus, our study provides new insights for understanding the mechanisms of biochar as an alternative to agricultural waste recycling and a method to promote environmental safety.",
"introduction": "1. Introduction The rhizosphere is an active and dynamic interface essential for the well-being of plants [ 1 , 2 ]. Plants take up water and nutrients from the rhizosphere, whereas their roots secrete a variety of compounds into the interface, causing changes in the physiochemical and biological properties of the surrounding bulk soil [ 3 ]. The corresponding differences between the rhizosphere and bulk soil are known as “rhizosphere effects” that play crucial roles in determining soil biogeochemical processes [ 4 ]. Despite the growing recognition regarding the overall rhizosphere effects on soil biogeochemistry [ 5 , 6 ], little is known about the rhizosphere effects on soil microbial genomes that are the base for soil nutrient cycling [ 7 ]. Biochar is regularly used to improve soil quality and potentially mitigate global change [ 8 , 9 ]. Straw biochar can promote crop productivity by not only enhancing the uptake of soil available resources but also driving soil microbiome [ 10 , 11 ]. Despite the enhancements of the metabolic potential and subsequent soil available resources after biochar application, the underlying mechanisms are yet unknown [ 12 , 13 ]. Therefore, it is imperative to explore the shift of microbiomes in the rhizosphere, which helps to understand the rhizosphere effects on soil nutrients in biochar-amended soils. The effects of biochar on the diversity and community composition of soil and rhizosphere microbiota were documented [ 14 ]. The underlying effects of biochar on the microbial metagenome, however, were not clear. The microbial metagenome was the most relevant part of the microbial functional traits in determining nutrient cycling, which might be involved in the biochar effects. For example, a meta-analysis revealed that biochar could increase the uptake of phosphorus for plants but the phosphorus cycling genes were not elusive [ 11 ]. It is also noted that the metagame approach provides a holistic way to analyze the microbial community without PCR bias [ 15 ], which might be a benefit for understanding both microbial taxonomy and functions [ 16 ]. Here, we conducted a field experiment to investigate the rhizosphere effects of wheat under straw biochar applications (See Supplementary Information for more details). Briefly, three treatments were set up in triplicates, including no straw application (control, CK), straw cut into 5 cm lengths and returned to the field (straw return, SR), and straw transformed to biochar and returned to the field (straw biochar return, SBR). Bulk and rhizosphere soil sampling, soil analyses, and metagenome sequencing followed [ 12 ]. The rhizosphere effects were quantified as the magnitude of differences between the rhizosphere and bulk soils relative to the bulk soil [ 4 ]. We hypothesized that straw biochar application would stimulate the rhizosphere effects in both soil available resources and soil microbiome, thus promoting the yields of wheat.",
"discussion": "4. Discussion Our results that straw biochar-driven amplification of rhizosphere effects on the diversity and community composition of the microbiome supported our hypothesis. Meanwhile, our results showed that straw biochar enhanced the rhizosphere effects on taxonomic and functional diversities in control and straw-returning treatments, with the net effects transferred from negative to positive. These results together explained the positive effects of biochar on the availability of soil resources, such as TN and AK, which showed the same trend in the present study. Biochar can increase soil microbial diversity and metabolic activities [ 12 , 28 ], thus resulting in positive effects on the diversity of both microbial taxa and functional genes. In addition, increases in the soil available resources might diversify the bacteria or archaea communities hosting and diversifying the virus communities [ 29 , 30 ]. These results highlight the comprehensive effects of biochar on the diversity of all components in soil biota. Simultaneously, the rhizosphere effects contributed to the major variation in community composition for the total gene, archaea, bacteria, and virus. Our results showed that soil resources and metabolism genes exhibited different responses to straw biochar application. In accordance, the positive rhizosphere effects occurred for the soil resources, but the negative took place for the metabolism genes. Typically, biochar directly functions as a nutrient source and indirectly alters the contents of soil nutrients for plant roots [ 31 ]. In the present study, the straw biochar increased the contents of AP, AK, and SOC by 20~50% in the rhizosphere, which presumably benefits plant growth [ 32 ]. However, biochar application resulted in negative rhizosphere effects on nitrogen gene abundance. This might be attributed to the significant increase in biochar-driven nitrogen genes in the bulk soils compared to the rhizosphere. Higher supplies of nutrients in biochar-amended soils might suppress the abundance of microbial metabolism genes [ 12 ]. Our results that straw return resulted in a decrease in wheat yields, while the straw biochar return increased the wheat yields, indicate that there is a great advantage for the straw biochar application in agricultural ecosystems in the form of waste straw recycling [ 33 ]. We also noted that the effects of straw biochar were mainly exerted on soil resources and not on the metabolism genes. This suggests that the straw biochar might serve as a resource pool providing resources for crops in the field. It is documented that biochar is valuable as a fertilizer when resource deficiency is a major constraint on crop productivity [ 34 ]. The pyrolysis step of biochar production produces more available resources, such as potassium, in the present study. We found that the rhizosphere effects of potassium were positive while other resources were negative for biochar returning treatment. Plant roots prefer to live in biochar-amended soils, as the rhizosphere contains more biochar particles than the bulk soil [ 31 ]. Thus, the greater content of available nutrients, i.e., potassium in the present study, would be taken up by the plants and, therefore, enhance plant performance including crop yields."
} | 2,048 |
25177315 | PMC4132294 | pmc | 5,994 | {
"abstract": "Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.",
"introduction": "Introduction Elucidation of metabolic and regulatory barriers in microbial conversion of lignocellulosic sugars to ethanol is crucial for both the immediate goal of economical cellulosic ethanol and for the long-term development of next-generation biofuels and sustainable chemicals from renewable biomass. Efficient conversion of lignocellulose (LC) hydrolysates is limited by multiple factors (Mills et al., 2009 ; Lau and Dale, 2010 ), including high osmolarity (Underwood et al., 2004 ; Purvis et al., 2005 ; Miller and Ingram, 2007 ), toxicity of the conversion products (Ingram and Buttke, 1984 ), and inhibitors of microbial metabolism and growth generated during the deconstruction of LC (Zaldivar et al., 1999 ; Wang et al., 2011a ; Tang et al., submitted). Understanding and overcoming the barriers created by LC-derived inhibitors presents significant challenges as their composition can vary depending on the biomass source of LC, the methods used to deconstruct the LC, and the diverse metabolic and regulatory responses of microbes to inhibitors (Klinke et al., 2004 ; Liu, 2011 ). Synergy among the inhibitors, the high osmolarity inherent to hydrolysates, and toxicity of conversion products (e.g., ethanol) are additional factors that contribute to the complex molecular landscape of lignocellulosic hydrolysates (Klinke et al., 2004 ; Liu, 2011 ; Piotrowski et al., 2014 ). Release of sugars from LC typically requires either acidic or alkaline treatment of biomass prior to or coupled with chemical or enzymatic hydrolysis (Chundawat et al., 2011 ). Acidic treatments generate significant microbial inhibitors by condensation reactions of sugars (e.g., furfural and 5-hydroxymethylfurfural). Microbes typically detoxify these aldehydes by reduction or oxidation to less toxic alcohols or acids (Booth et al., 2003 ; Herring and Blattner, 2004 ; Marx et al., 2004 ; Jarboe, 2011 ), but these conversions also directly or indirectly consume energy that otherwise would be available for biofuel synthesis (Miller et al., 2009a , b ) The impact of these inhibitors is especially significant for C5 sugars like xylose whose catabolism provide slightly less cellular energy (Lawford and Rousseau, 1995 ), and can be partially ameliorated by replacing NADPH-consuming enzymes with NADH-consuming enzymes (Wang et al., 2013 ). Alkaline treatments, for instance with ammonia, are potentially advantageous in generating fewer toxic aldehydes, but the spectrum of inhibitors generated by alkaline treatments is less well characterized and their effects on microbial metabolism are less well understood. We have developed an approach to elucidate the metabolic and regulatory barriers to microbial conversion in LC hydrolysates using ammonia fiber expansion (AFEX) of corn stover, enzymatic hydrolysis, and a model ethanologen (GLBRCE1) engineered from the well-studied bacterium E. coli K-12 (Schwalbach et al., 2012 ). Our strategy is to compare anaerobic metabolic and regulatory responses of the ethanologen in authentic AFEX-pretreated corn stover hydrolysate (ACSH) to responses to synthetic hydrolysates (SynHs) designed to mimic ACSH with a chemically defined medium. GLBRCE1 metabolizes ACSH in exponential, transition, and stationary phases but, unlike growth in traditional rich media (Sezonov et al., 2007 ), GLBRCE1 enters stationary phase (ceases growth) long before depletion of available glucose but coincident with exhaustion of amino acid sources of organic nitrogen (Schwalbach et al., 2012 ). The growth-arrested cells remain metabolically active and convert the remaining glucose, but not xylose, into ethanol (Schwalbach et al., 2012 ). Our first version of SynH (SynH1) matched ACSH for levels of glucose, xylose, amino acids, and some inorganics, overall osmolality, and the amino-acid-dependent growth arrest of GLBRCE1 (Schwalbach et al., 2012 ). However, gene expression profiling revealed that SynH1 cells experienced significant osmotic stress relative to ACSH cells, whereas ACSH cells exhibited elevated expression of efflux pumps, notably of aaeAB that acts on aromatic carboxylates (Van Dyk et al., 2004 ), relative to SynH1 cells (Schwalbach et al., 2012 ). Osmolytes found in ACSH (betaine, choline, and carnitine) likely explained the lower osmotic stress, whereas phenolic carboxylates derived from LC (e.g., coumarate and ferulate) likely explained efflux pump induction possibly via the AaeR and MarA/SoxS/Rob regulons known to be induced by phenolic carboxylates (Sulavik et al., 1995 ; Dalrymple and Swadling, 1997 ). We also observed elevated expression of psp, ibp , and srl genes associated with ethanol stress at ethanol concentrations three-fold lower than previously reported to induce expression (Yomano et al., 1998 ; Goodarzi et al., 2010 ) and thus consistent with a synergistic stress response with the LC-derived inhibitors. These findings led us to hypothesize that the collective effects of osmotic, ethanol, and LC-derived inhibitor stresses created an increased need for ATP and reducing equivalents that was partially offset in early growth phase by catabolism of amino acids, as N and possibly S sources. However, as these amino acids are depleted, cells transition to stationary phase where they continue to catabolize glucose for maintenance ATP and NAD(P)H but are unable to generate sufficient energy for cell growth or efficient xylose catabolism. To test this hypothesis, we developed a new SynH formulation (SynH2) that faithfully replicates the physiological responses in ACSH and the effects of LC-derived inhibitors. Using SynH2 with and without the LC-derived inhibitors, we generated and analyzed metabolomic, gene expression, and proteomic data to define the effects of inhibitors on bacterial gene expression and physiology. The analysis allowed identification of key regulators that may provoke stress responses in the presence of LC-derived inhibitors and suggest that coping mechanisms employed by E. coli to deal with lignocellulosic stress drains cellular energy, thus limiting xylose conversion.",
"discussion": "Discussion Results of our investigation into the effects of LC-derived inhibitors on E. coli ethanologenesis support several key conclusions that will guide future work. First, a chemically defined mimic of ACSH (SynH2) that contained the major inhibitors found by chemical analysis of ACSH adequately replicated both growth and the rates of glucose and xylose conversion to ethanol by E. coli . SynH2-replication of ACSH required inclusion of osmolytes found in ACSH and established that, at the ratios present in ACSH, phenolic carboxylates and amides, which are not metabolized by E. coli , had a greater overall impact on cell growth than phenolic aldehydes and furfurals, which were metabolized. In both SynH2 and ACSH, E. coli entered a metabolically active stationary phase as cells exhausted organic sources of N and S (e.g., amino acids) and during which the inhibitors greatly reduced xylose conversion. The impact of inhibitors on cellular energetics reduced levels of ATP, NADH, and NADPH and was seen most dramatically for energetically challenging processes requiring NADPH (like SO −2 4 assimilation and deoxyribonucleotide production), during transition to the stationary phase on ATP-dependent NH 3 assimilation, and in elevated pyruvate levels presumably reflecting reduced NADH-dependent flux of pyruvate to ethanol (Figure 7 ). The direct effects of the inhibitors on cells appear to be principally mediated by transcriptional rather than translational regulators, with the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC being the most prominent players. Although the effect of the inhibitors on transcriptional regulation of the efflux pumps was striking, increased efflux activity itself may perturb cellular metabolism. For example, Dhamdhere and Zgurskaya ( 2010 ) have shown that deletion of the AcrAB-TolC complex results in metabolic shutdown and high NADH/NAD + ratios. By analogy, overexpression of efflux pumps may have the opposite effect (e.g., lowering of NADH/NAD + ratios), which is consistent with observations in this study. In addition, recent work suggests that the acrAB promoter is upregulated in response to certain cellular metabolites (including those related to cysteine and purine biosynthesis), which are normally effluxed by this pump (Ruiz and Levy, 2014 ). Therefore, upregulation of AcrAB-TolC may impact homeostatic mechanisms of cellular biosynthetic pathways, resulting in continuous upregulation of pathways that require large amounts of reducing power in the form of NADPH. It is also possible that LC-derived inhibitors perturb metabolism directly in ways that generate additional AcrAB-TolC substrates, potentially increasing energy-consuming efflux further. Given these intricacies, further studies to unravel the mechanistic details of the effects of efflux pump activity on cellular metabolism, as a result of exposure to LC-derived inhibitors, are warranted. Figure 7 Major Regulatory responses of E. coli to aromatic inhibitors found in ACSH . The major E. coli responses to phenolic carboxylates and amides (left) or responses to aldehydes (right) are depicted. Green panels , regulators and signaling interactions that mediate the regulatory responses. Pink panels , direct targets of the regulators that consume reductant (NADPH) for detoxification reactions or deplete the proton motive force through continuous antiporter efflux of aromatic carboxylates. Blue panels , indirect effects of inhibitors mediated by reductions in ATP and NADPH levels. The inability of cells to convert xylose in the presence of inhibitors appears to result from a combination of both effects on gene expression and some additional effect on transport or metabolism. The inhibitors lowered xylose gene expression (XylR regulon; xylABFGH ) by a factor of 3-5 during all three growth phases (Table S4 ). This effect was not caused by the previously documented AraC repression (Desai and Rao, 2010 ), since it persisted in SynH2 when we replaced the AraC effector L-arabinose with D-arabinose, but might reflect lower levels of cAMP caused by the inhibitors (Figure 4 ); both the xylAB and xylFGH operons are also regulated by CRP·cAMP. Nonetheless, significant levels of XylA, B, and F were detected even in the presence of inhibitors (Table S7D ), even though xylose conversion remained inhibited even after glucose depletion (Table 2 ). Thus, the inability to convert xylose may also reflect either the overall impact of inhibitors on cellular energetics somehow making xylose conversion unfavorable or an effect of xylose transport or metabolism that remains to be discovered. Further studies of the impact of inhibitors on xylose transport and metabolism are warranted. It would be particularly interesting to test SynH formulations designed to compare the conversion efficiencies of xylose, arabinose, and C6 sugars other than glucose. The central focus of this study was to understand the impact of inhibitors of gene expression regulatory networks. The apparent lack of involvement of post-transcriptional regulation suggests that E. coli mounts a defense against LC-derived inhibitors principally by controlling gene transcription, probably reflecting evolution of specific bacterial responses to LC-derived inhibitors. Although enteric bacteria do not ordinarily encounter industrial lignocellulosic hydrolysates, they likely encounter the same suite of compounds from digested plant material in the mammalian gut. Thus, evolution of specific responses is reasonable. A key question for future studies is whether phenolic amides, not ordinarily present in digested biomass, will also invoke these responses in the absence of carboxylates or aldehydes. We note that the apparent absence of a translational regulatory response in the cellular defense against LC-derived inhibitors does not preclude involvement of either direct or indirect post-transcriptional regulation in fine-tuning the response. Our proteomic measurements would likely not have detected fine-tuning. Additionally, we did detect an apparently indirect induction by inhibitors of protein degradation in stationary phase, possibly in response to C starvation (Figure 6C ). Finally, we note that the sRNA micF, a known post-transcriptional regulator, is a constituent of the MarA/SoxS/Rob regulon and was upregulated by inhibitors. Although confidence was insignificant due to poor detection of sRNAs in RNAseq data, the induction of micF was confirmed in a separate study of sRNAs (Ong and Landick, in preparation). Thus, a more focused study of the involvement of sRNAs in responses to LC inhibitors would likely be informative. MarA/SoxS/Rob is a complex regulon consisting of the three inter-connected primary AraC-class regulators that bind as monomers to 20-bp sites in promoters with highly overlapping specificity and synergistically regulate ~50 genes implicated in resistance to multiple antibiotics and xenobiotics, solvent tolerance, outer membrane permeability, DNA repair, and other functions (Chubiz et al., 2012 ; Duval and Lister, 2013 ; Garcia-Bernardo and Dunlop, 2013 ) (Figure 7 ). Twenty-three genes, including those encoding the AcrAB·TolC efflux pump, the NfsAB nitroreductases, the micF sRNA, superoxide dismutase, some metabolic enzymes (e.g., Zwf, AcnA, and FumC) and incompletely characterized stress proteins are controlled by all three regulators, whereas other genes are annotated as being controlled by only a subset of the regulators (Duval and Lister, 2013 ), www.ecocyc.org ; (Keseler et al., 2013 ). MarA and SoxS lack the C-terminal dimerization domain of AraC; this domain is present on Rob and appears to mediate regulation by aggregation that can be reversed by effectors (Griffith et al., 2009 ). Inputs capable of inducing these genes, either through the MarR and SoxR repressors that control MarA and SoxS, respectively, or by direct effects on Rob include phenolic carboxylates, Cu 2+ , a variety of organic oxidants, dipyridyl, decanoate, bile salts, Fis, and Crp·cAMP (Martin and Rosner, 1997 ; Rosner et al., 2002 ; Rosenberg et al., 2003 ; Chubiz and Rao, 2010 ; Duval and Lister, 2013 ; Hao et al., 2014 ) (Figure 7 ). Given these diverse inputs, it seems highly likely that ferulate and coumarate in ACSH induce the MarA/SoxS/Rob regulon via MarR. Indeed, LC-hydrolysate and ferulate induction of MarA has been reported (Lee et al., 2012 ). Interestingly, Cu 2+ recently was shown to induce MarR by oxidation to create MarR disulfide dimer (Hao et al., 2014 ). Given the elevated levels of Cu 2+ in ACSH reflected by induction of Cu 2+ efflux (Figure 2 ; Table S4 ), induction of MarA/SoxS/Rob in ACSH may result from synergistic effects of Cu 2+ and phenolic carboxylates, oxidants that affect SoxR, and yet-to-be-determined compounds that affect Rob. A second response in LC-derived inhibitors appears to be mounted by the LysR-type regulator AaeR, which controls the AaeAB aromatic carboxylate efflux system (Van Dyk et al., 2004 ) (Figure 7 ). Both phenolic and aryl carboxylates induce AaeAB through AaeR, but little is known about its substrate specificity or mechanism of activation. Two distinct regulators, YqhC and FrmR, control synthesis of the YqhD/DkgA NAPDH-dependent aldehyde reductases and the FrmAB formaldehyde oxidase, respectively (Herring and Blattner, 2004 ; Turner et al., 2011 ). Even less is known about these regulators, although the DNA-binding properties of YqhC have been determined. In particular, it is unclear how aldehydes cause induction, although the current evidence suggests effects on YqhC are likely to be indirect. Given the central role of the regulators AaeR, YqhC, and FrmR in the cellular response to LC-derived inhibitors, further study of their properties and mechanisms is likely to be profitable. With sufficient understanding and engineering, they could be used as response regulators to engineer cells that respond to LC-inhibitors in ways that maximize microbial conversion of sugars to biofuels. What types of responses would optimize biofuel synthesis? It appears the naturally evolved responses, namely induction of efflux systems and NADPH-dependent detoxification pathways, may not be optimal for efficient synthesis of biofuels. We infer this conclusion for several reasons. First, our gene expression results reveal that crucial pathways for cellular biosynthesis that are among the most energetically challenging processes in cells, S assimilation, N assimilation, and ribonucleotide reduction, are highly induced by LC-derived inhibitors (Figures 2 , 7 ; Table S4 ). A reasonable conjecture is that the diversion of energy pools, including NADPH and ATP, to detoxification makes S assimilation, N assimilation, and ribonucleotide reduction difficult, increasing expression of genes for these pathways indirectly. The continued presence of the phenolic carboxylates and amides (Figure 3 ) likely causes futile cycles of efflux. As both the AcrAB and AaeAB efflux pumps function as proton antiporters (Figure 7 ), continuous efflux is expected to decrease ATP synthesis by depleting the proton-motive force. Although this response makes sense evolutionarily because it protects DNA from damage by xenobiotics, it does not necessarily aid conversion of sugars to biofuels. Disabling these efflux and detoxification systems, especially during stationary phase when cell growth is no longer necessary, could improve rates of ethanologenesis. Indeed, Ingram and colleagues have shown that disabling the NADPH-dependent YqhD/DkgA enzymes or better yet replacing them with NADH-dependent aldehyde reductases (e.g., FucO) can improve ethanologenesis in furfural-containing hydrolysates of acid-pretreated biomass (Wang et al., 2011a , 2013 ). That simply deleting yqhD improves ethanologenesis argues that, in at least some cases, it is better to expose cells to LC-derived inhibitors than to spend energy detoxifying the inhibitors. Some previous efforts to engineer cells for improved biofuel synthesis have focused on overexpression of selected efflux pumps to reduce the toxic effects of biofuel products (Dunlop et al., 2011 ). Although this strategy may help cells cope with the effects of biofuel products, our results suggest an added potential issue when dealing with real hydrolysates, namely that efflux pumps may also reduce the rates of biofuel yields by futile cycling of LC-derived inhibitors. Thus, effective use of efflux pumps will require careful control of their synthesis (Harrison and Dunlop, 2012 ). An alternative strategy to cope with LC-derived inhibitors may be to devise metabolic routes to assimilate them into cellular metabolism. In conclusion, our findings illustrate the utility of using chemically defined mimics of biomass hydrolysates for genome-scale study of microbial biofuel synthesis as a strategy to identify barriers to biofuel synthesis. By identifying the main inhibitors present in ammonia-pretreated biomass hydrolysate and using these inhibitors in a synthetic hydrolysate, we were able to identify the key regulators responsible for the cellular responses that reduced the rate of ethanol production and limited xylose conversion to ethanol. Knowledge of these regulators will enable design of new control circuits to improve microbial biofuel production. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."
} | 5,388 |
26861023 | PMC4752607 | pmc | 5,995 | {
"abstract": "ABSTRACT The genomes of many photosynthetic and nonphotosynthetic bacteria encode numerous phytochrome superfamily photoreceptors whose functions and interactions are largely unknown. Cyanobacterial genomes encode particularly large numbers of phytochrome superfamily members called cyanobacteriochromes. These have diverse light color-sensing abilities, and their functions and interactions are just beginning to be understood. One of the best characterized of these functions is the regulation of photosynthetic light-harvesting antenna composition in the cyanobacterium Fremyella diplosiphon by the cyanobacteriochrome RcaE in response to red and green light, a process known as chromatic acclimation. We have identified a new cyanobacteriochrome named DpxA that maximally senses teal (absorption maximum, 494 nm) and yellow (absorption maximum, 568 nm) light and represses the accumulation of a key light-harvesting protein called phycoerythrin, which is also regulated by RcaE during chromatic acclimation. Like RcaE, DpxA is a two-component system kinase, although these two photoreceptors can influence phycoerythrin expression through different signaling pathways. The peak responsiveness of DpxA to teal and yellow light provides highly refined color discrimination in the green spectral region, which provides important wavelengths for photosynthetic light harvesting in cyanobacteria. These results redefine chromatic acclimation in cyanobacteria and demonstrate that cyanobacteriochromes can coordinately impart sophisticated light color sensing across the visible spectrum to regulate important photosynthetic acclimation processes.",
"introduction": "INTRODUCTION The phytochrome superfamily is an important group of photoreceptors whose members are widely distributed in photosynthetic and nonphotosynthetic prokaryotes and eukaryotes ( 1 – 3 ). These proteins were first identified and characterized in land plants, where they are known to control a number of light-dependent processes ( 1 , 4 – 7 ). Phytochrome-class proteins are dual color light switches that contain one or more light-sensing bilin chromophores, each covalently attached to the protein at conserved cysteine residues. Every bilin chromophore found in phytochromes can exist in one of two forms, each of which maximally absorbs a different wavelength of light. The structural change from one form to the other is most effectively driven by the chromophore’s absorption of a photon of light. Switching between forms alters the structure of its attached protein in a unique way ( 8 ), leading to changes in how specific physiological processes are regulated. Although all known phytochromes in plants and nonphotosynthetic bacteria isomerize between red light- and far-red light-absorbing forms ( 3 , 4 , 6 , 7 ), phytochrome-like photoreceptors in large numbers of other species sense many additional wavelengths of light. The numbers, diversity, and range of light color-sensing capabilities of phytochrome superfamily members are the greatest in cyanobacteria and algae, with the filamentous cyanobacterium Fremyella diplosiphon containing as many as 27 different family members ( 9 – 11 ). In cyanobacteria, this variety is primarily due to the presence of a large number of cyanobacteriochromes, a subgroup within the phytochrome superfamily that uses only a GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain for light color sensing ( 12 ). A major unanswered question is how cyanobacteria actually use this capacity to respond to so many different light colors and how cross talk between photoreceptors is controlled. A well-studied response that is regulated by a cyanobacteriochrome is type III chromatic acclimation (CA3), a process which involves changes in the composition of the photosynthetic light-harvesting antennae and is controlled by a cyanobacteriochrome called RcaE ( 13 – 15 ). In the CA3 model organism F. diplosiphon , RcaE directs a signal transduction pathway that in red light represses synthesis of the red-colored protein phycoerythrin and activates production of the blue-green-colored protein phycocyanin. In green light, RcaE does not repress phycoerythrin production or activate phycocyanin synthesis, resulting in an increased phycoerythrin-to-phycocyanin ratio. Consequently, because phycoerythrin and phycocyanin are major components of the photosynthetic light-harvesting antennae, which are called phycobilisomes, these changes result in a dramatic, reversible change in cell color phenotype, from blue-green in red light to brick-red in green light ( 16 ). Since phycoerythrin best absorbs green light and phycocyanin most effectively absorbs red light, CA3 provides a fitness advantage in changing light color environments by increasing the efficiency of photon capture for photosynthesis ( 17 ). RcaE is also a histidine kinase, switching between the kinase-active green-absorbing form (RcaE g ) in red light and the kinase-inactive red-absorbing form (RcaE r ) in green light ( 15 ). In red light, RcaE g phosphorylates a complex two-component system comprised of the single domain response regulator RcaF and complex response regulator/transcription factor RcaC. This represses transcription of cpeCDESTR (here cpeC ) and cpeBA , operons required to produce phycoerythrin-containing phycobilisomes, and activates transcription of the phycocyanin-encoding operon cpcB2A2 ( 18 , 19 ). There are only single copies of cpeC and cpeBA in the F. diplosiphon genome. Here, we demonstrate that a previously uncharacterized cyanobacteriochrome named DpxA provides an additional level of photoreceptor-based regulation of phycoerythrin expression. DpxA maximally responds to teal and yellow light and regulates phycoerythrin abundance at least in part through a signal transduction pathway that is independent of the Rca system. Our data indicate that DpxA represses accumulation of phycoerythrin in yellow light and does not repress it in blue light. The absorption maxima of DpxA precisely flank the absorption maximum of RcaE g , allowing F. diplosiphon to use a second cyanobacteriochrome to fine-tune the RcaE regulation of phycoerythrin levels in the blue-to-yellow region of the light spectrum. These findings reveal how multiple cyanobacteriochromes are used by cyanobacteria to provide sophisticated light color sensing for the control of photosynthetic light-harvesting gene expression across the visible spectrum.",
"discussion": "DISCUSSION The capacity of DpxA and RcaE to respond to different regions of the light spectrum allows F. diplosiphon to actively regulate the abundance of phycoerythrin in almost all light colors and provides a new, previously unrecognized degree of sophistication in the regulation of photosynthetic gene expression by light color ( Fig. 5 ). The influence of the DpxA and RcaE systems is evident across the visible spectrum. In red light, RcaE is in its active form and the influence of the Rca system is strong, activating phycocyanin expression and repressing phycoerythrin expression. The effect of DpxA in red light is minor ( Fig. 3D ), perhaps because DpxA is an approximately equimolar mixture of active and inactive forms in this light color ( Fig. 4 ). Shifting cells to yellow light, the DpxA system is the predominant means of repressing phycoerythrin accumulation, as RcaE has largely converted into its inactive form ( Fig. 4 ). In the blue region of the spectrum, both RcaE and DpxA are in their inactive states and the repression of phycoerythrin abundance by these two photoreceptors is at its minimum ( Fig. 4 and 5 ). This may be a mechanism through which the cells fully commit to phycoerythrin accumulation only when the wavelengths of light between red and green are no longer available for photosynthesis. The possible existence of a color-sensing mechanism capable of fine-tuning light-harvesting biogenesis in the green region of the spectrum such as the one that we describe here has been previously suggested but not identified until now ( 10 ). DpxA responds to a narrower range of wavelengths than the well-characterized cyanobacteriochrome RcaE ( 15 ) ( Fig. 5 ), and although both repress phycoerythrin, DpxA does not activate phycocyanin in wild-type cells ( Fig. 1B ). Thus, DpxA functions as a fine-tuning modifier of phycobilisome biogenesis, affecting phycoerythrin accumulation in response to a subset of light colors within the green range, a spectral region in which RcaE g absorbs broadly and thus is not useful for fine color discrimination and where phycocyanin regulation is less relevant. Cyanobacteriochrome family members in other cyanobacterial species vary in the range of wavelengths to which they respond ( 10 ). Thus, the integration of the DpxA and RcaE color-sensing systems in the control of chromatic acclimation may be an indication of similar systems yet to be discovered in other organisms. Other regulatory systems that influence phycoerythrin abundance exclusively have been described in F. diplosiphon and related species. The Cgi pathway has been shown to repress phycoerythrin accumulation in red light through a posttranscription initiation mechanism ( 22 , 23 ), and a translation initiation factor 3 named IF3α has been identified in the light color-mediated repression of phycoerythrin expression in F. diplosiphon ( 24 ). Interactions between DpxA, the Cgi system, and IF3α have not yet been discovered. During type II chromatic acclimation (CA2) in Nostoc punctiforme , phycoerythrin is also repressed in red light, using the green-red-sensing cyanobacteriochrome CcaS ( 25 , 26 ). It was postulated that CcaS may be similar to the sensor for the Cgi system. However, DpxA controls a teal-yellow-sensing system, distinguishing it from the green-red-sensing CcaS-controlled pathway. Furthermore, despite the existence of DpxA homologs with 67% or greater sequence identity in 15 other cyanobacterial species (see Fig. S5 in the supplemental material), DpxA has only 28% sequence identity with full-length CcaS. The DpxA system therefore appears to be independent of the RcaE-controlled portion of the CA3 system, the CA2 system, and perhaps the Cgi system, and is a previously unrecognized mechanism for acclimation of phycoerythrin in the green light range. The identification of DpxA as a repressor of phycoerythrin abundance makes it the first yellow-light-sensing cyanobacteriochrome with a known physiological role. DpxA-like proteins must be broadly important for light sensing in cyanobacteria, since a large number of predicted photoreceptors with strong sequence relatedness (greater than or equal to 67% identity) to the entire length of DpxA exist in many cyanobacteria (see Fig. S5 in the supplemental material). The large number of phytochrome-class photoreceptors in F. diplosiphon also raises the possibility that another cyanobacteriochrome plays a role for RcaE r that is similar to the role DpxA plays for RcaE g , fine-tuning phycocyanin abundance in the red light region of the spectrum. If so, we would expect the absorption maxima for such a photoreceptor to approximately evenly flank the 661-nm absorption maximum of RcaE r . The independent color-sensing capabilities of DpxA and RcaE, combined with the overlapping roles of these two photoreceptors, provide an important new perspective on how cyanobacteriochromes operate in the natural environment, where light is not monochromatic. Chromatic acclimation was first described in Oscillatoria sancta as a dramatic switch of cell color from red when grown in green light to blue-green when grown in red light ( 27 ). Since then, other cyanobacteria have been shown to change pigmentation in response to two light colors ( 16 , 22 , 28 – 30 ). Our discovery that CA3 in F. diplosiphon is controlled by two sensing systems that track four different light colors demonstrates that this process is simultaneously responding to many different ratios of light color in the environment. The presence of these two acclimation systems in one organism also underscores the value of fine-tuning the composition of the photosynthetic light-harvesting pigments to match subtle changes that occur in the spectral distribution of ambient light color. Recent studies have shown that multiple cyanobacteriochromes can coordinately regulate phototaxis and aggregation ( 31 , 32 ). However, the absorption characteristics of the photoreceptors involved in these processes are similar (UV/blue for phototaxis and blue/green for aggregation). Therefore, the physiological role of using more than one photoreceptor in these systems does not appear to be to significantly expand the range of light color sensing. It is possible that the photoreceptors in these systems serve intensity-sensing roles, which could be conferred by different reversion kinetics and/or output functions for each of the photoreceptors. The results presented here provide a novel example of how cyanobacteriochromes interact by demonstrating that DpxA and RcaE have very different light absorption properties yet share overlapping functional roles in the regulation of phycoerythrin abundance. The gradual, coordinated release of the tandem repressing functions of these two photoreceptors as cells transition from red to blue light, especially through the yellow/teal region of the spectrum, demonstrates a much higher level of sophistication in the light color regulation of phycobilisome biogenesis than initially believed ( 16 , 27 – 29 , 33 ). In fact, it is not entirely surprising that such precise regulation exists, since efficient light harvesting is critical for cyanobacterial fitness under changing light conditions ( 17 ) and the production of these antennae, which may comprise up to 60% of the total soluble protein in a cyanobacterial cell ( 33 ), represents a major investment of resources for these organisms."
} | 3,476 |
24693254 | PMC3943290 | pmc | 5,997 | {
"abstract": "Obligate aerobic AMF taxa have high species richness under waterlogged conditions, but their ecological role remains unclear. Here we focused on AM fungal mediation of plant interactions in a marshland plant community. Five cooccurring plant species were chosen for a neighbor removal experiment in which benomyl was used to suppress AMF colonization. A Phragmites australis removal experiment was also performed to study its role in promoting AMF colonization by increasing rhizosphere oxygen concentration. Mycorrhizal fungal effects on plant interactions were different for dominant and subdominant plant species. AMF colonization has driven positive neighbor effects for three subdominant plant species including Kummerowia striata , Leonurus artemisia , and Ixeris polycephala . In contrast, AMF colonization enhanced the negative effects of neighbors on the dominant Conyza canadensis and had no significant impact on the neighbor interaction to the dominant Polygonum pubescens . AM colonization was positively related to oxygen concentration. P. australis increased oxygen concentration, enhanced AMF colonization, and was thus indirectly capable of influencing plant interactions. Aerobic AM fungi appear to be ecologically relevant in this wetland ecosystem. They drive positive neighbor interactions for subdominant plant species, effectively increasing plant diversity. We suggest, therefore, that AM fungi may be ecologically important even under waterlogged conditions.",
"introduction": "1. Introduction The arbuscular mycorrhiza, which is a mutualistic symbiosis between plants and arbuscular mycorrhizal (AM) fungi, may enhance plant nutrient acquisition, protect host plants from abiotic (e.g., drought) and biotic (e.g., pathogen) stresses, and mediate plant-plant interactions [ 1 ]. The majority of ecological studies on arbuscular mycorrhizas have concentrated on the distribution (~80% of land plant species) and the role of AM fungi in terrestrial ecosystems, as the fungi are considered to be obligate aerobes [ 2 , 3 ]. Waterlogged soils are generally anoxic [ 4 ] and plants under such conditions have traditionally been regarded as nonmycorrhizal [ 5 ]. However, mycorrhizal colonization has been reported in waterlogged plants [ 6 – 9 ]. For example, AM colonization does not seem to be significantly disrupted during short-term waterlogging events [ 10 ]. Although flooding [ 8 ] and redox potential values [ 11 ] can influence AM spore numbers and mycorrhizal colonization, molecular technique revealed that aquatic plants may harbor as high an AM fungal species richness as terrestrial plants [ 8 ]. However, despite the examination of the existence or prevalence of arbuscular mycorrhiza fungi under waterlogged conditions, more information regarding the ecological significance of AMF in this habitat is still very much needed. In this study we focused on arbuscular mycorrhizal fungal mediation of plant-plant interactions under waterlogged conditions. Plant-plant interactions are important in driving plant population dynamics [ 12 ], plant community structure [ 13 ], and ecosystem functions [ 14 ]. Beside the long concerned resource competition (negative interaction), the so-called “nurse plant” may benefit the performance of neighboring plant (i.e., positive interaction), through the accumulation of nutrients, provision of shade, amelioration of disturbance, or protection from herbivores [ 15 ]. The outcome of plant-plant interactions reflects the balance of negative and positive effects acting simultaneously [ 16 ], and the inability to predict the nature of species interactions under various environmental contexts is a major gap in our ecological understanding [ 17 ]. Plant interactions are known to be influenced by both abiotic (e.g., the stress gradient hypothesis) [ 18 ] and biotic (e.g., herbivores and mycorrhizal fungi) [ 19 , 20 ] factors. Because of their widespread distribution, fungal mediation of plant interactions by mycorrhizal fungi is of increasing interest. There is a great deal of variation in the magnitude and direction of the effects of mycorrhizal fungi on plant-plant interactions. In a review paper, van der Heijden and Horton [ 20 ] proposed that the mycorrhizal network is very important in ameliorating competition in natural ecosystems. Mycorrhizal fungi also tended to reduce plant competition under saline conditions [ 21 ] or enhance positive neighbor effects in severe drought [ 22 ]. On the other hand, other studies have shown that mycorrhizal fungi increase plant competition [ 23 – 27 ]. For example, mycorrhizal networks were found to amplify size inequality which was originated from intraspecific competition [ 26 , 27 ]. Although AMF are not generally characterized as being host specific, AMF species can display host preferences [ 28 – 31 ]. The effect of AM mutualism ranges along a mutualism-parasitism continuum depending on plant species, the life history, and ecological conditions [ 32 , 33 ]. Then AMF alter plant community structure by affecting the relative abundance of plant species and plant-species diversity [ 34 – 37 ]. AMF could promote plant coexistence by increasing the competitive ability of less competitive species [ 38 , 39 ] or reduce coexistence by reinforcing competitive dominance of the dominant plant species [ 40 ]. In wetland systems, Wolfe et al. [ 41 ] and Daleo et al. [ 42 , 43 ] show that marsh plant zonation and community structure may be dependent on mycorrhizal fungi in these wetland systems. But we still need more studies to obtain a better understanding on mycorrhizal effects in wetland systems. Here we chose five cooccurring species differing in their competitive ability and environmental optima to evaluate possible AM fungal mediation of neighbor effects in marshland plant community. We also asked whether AM fungal mediation on plant interactions was dependent on oxygen concentration. AM fungal spores are usually abundant in waterlogged ecosystems but fail to develop because of these stressful conditions [ 8 ]. A field experiment at the Mar Chiquita coastal lagoon in Argentina demonstrates that fungal colonization is dependent on crab burrowing that can oxygenate soils [ 43 ]. Aerenchyma formation in aquatic macrophytes is one of the most obvious adaptive plant responses to flooding [ 44 ]. A well-developed aerenchyma in a plant would ensure an efficient exchange of gases between the atmosphere and the soil environment, and some of the oxygen transported through the aerenchyma may leak out of root pores into the surrounding soil [ 45 ]. The resulting thick layer of oxygenated soil around individual roots may maintain aerobic microbes. Then these plants may play a “nursing” role on the neighbor individuals with supporting aerobic, beneficial mycorrhizal fungi. Along the Yangtze River in China, marshland is characterized by the dominant plant species, Phragmites australis , a large perennial grass commonly found in wetlands. Vegetative organs of Phragmites australis have advanced aerenchyma [ 46 ], with two field neighbor removal experiments. We tested the hypothesis that (1) AM fungal symbiosis may show host preference in plant growth promotion and neighbor interaction among the five chosen plant species and (2) P. australis existence promotes AM fungal colonization of marsh plant roots by oxygenating waterlogged soils and, in turn, this interaction positively affects the AM fungal mediation of plant interactions.",
"discussion": "4. Discussion 4.1. Effects of Fungicide Benomyl application suppressed AM fungal colonization. Although some experiments have shown that pathogenic fungi [ 55 ] and other soil organisms such as root-feeding nematodes [ 56 ] can also be affected by benomyl, others reported that benomyl application had little or no effect on nonmycorrhizal plant and bacterial communities [ 42 ]. This is supported by the observation that benomyl application caused no difference in plant growth compared to pasteurized soil with an other soil microflora added back [ 57 ]. Because there is no method that only allows the elimination of AMF in a field setting, benomyl application may be one of the best options to suppress AMF in the field compared to other methods [ 43 , 57 ]. If benomyl affects pathogenic more than mycorrhizal fungi, plant growth should be promoted, not suppressed [ 57 ]. We have previously shown that the benomyl effect on Medicago sativa L. was mainly due to suppressing mycorrhizal colonization [ 21 , 58 ]. Here we made soil nutrient analysis with soil enzyme activity and culturable fungal unit measurement. We are confident that our results are actually due to AMF suppression as we found that benomyl application did not have significant effect on soil total nitrogen and mineralizable N, total P and available P, soil urease activity, acid phosphomonoesterase activity, and culturable fungal unit (see supporting information). These results suggest minimal experimental artefacts of benomyl application. Benomyl application led to a much reduced mycorrhizal colonization and decreased plant growth of the three subdominant plant species. Benomyl application did not affect growth of the two dominant plant species, suggesting that the dominant species are less dependent on mycorrhizal colonization than the subdominants. 4.2. AM Fungi and Plant Interactions under Waterlogged Conditions Plant growth may be either dependent or not dependent on mycorrhizal colonization, and AM fungal colonization and variation in AM fungal taxa both may alter interactions among plants in a variety of plant communities [ 20 , 38 , 57 , 59 – 62 ]. To our knowledge, only a few studies have been concerned with the role of AM fungi in plant communities experiencing waterlogging. Daleo et al. [ 42 ] reported that mycorrhizal fungi influenced interactions between Spartina densiflora and S. alterniflora and affected salt-marsh plant community structure. Here we showed that AM fungal colonization is an important contributor to plant growth and neighbor interactions in a plant community experiencing seasonally waterlogged, anaerobic conditions. Three of the five plant species, K. striata, L. Artemisia , and I. polycephala , showed growth dependence on mycorrhizal colonization. Facilitative neighbor effect on these three species was enhanced by mycorrhizal colonization, while mycorrhizal colonization on C. canadensis enhanced the competitive neighbor effect. Recently studies showed that cooccurring species with different stress tolerance and ecological optima may show differential responses to the same neighbors in a given community [ 49 ]. For example, the magnitude of positive neighbor effects among species was negatively correlated with the density of target plant species in an alpine meadow of the Qing-Hai Tibet Plateau [ 63 ]. Choler et al. [ 64 ] also showed negative neighbor effects on the target plants in the most favorable part of the niche and positive interactions in its most constrained part. Here we show that type (competitive or facilitative) of interspecific neighbor effect was dependent on species when the plant community was waterlogged; neighbor effects were negative or neutral for dominant plant species and facilitative for subdominant plant species. These species-specific neighbor effects were mainly driven by AMF. In this study we demonstrated that plant species vary in the degree to which they respond to AM fungi and plant neighbors. The dominant species P. pubescens and C. canadensis exhibited neutral or negative response to AMF and plant neighbors, while the three subdominant species exhibit positive responses to AMF and plant neighbors. The species-specific responsiveness to AMF as a mechanism in which AM fungi influenced plant community structure was first proposed by Bergelson and Crawley [ 65 ]. van der Heijden [ 66 ] suggested that the number and relative abundance of mycorrhizal-dependent plant species in the species pool can be used to predict how AM fungi affect communities. Here the ability of the three subdominant plant species to coexist with other plant species could therefore be highly dependent on AM fungal symbiosis. In contrast, C. canadensis was negatively affected by AM symbiosis and P. pubescens would not be directly affected by AM fungi. This high dependence of subdominant plant species on mycorrhiza has been proved to maintain high plant species richness and diversity [ 67 ]. It is interesting to note the asymmetry in the delivery of benefit between plant and AM fungi; the two dominant plant species maintain high mycorrhizal colonization but apparently receive little or no growth promotion, while growth and neighbor effects of subdominant plant species were promoted by reduced mycorrhizal colonization. The AM symbiosis may be largely nonspecific, but the extent of plant growth promotion by AM fungi and plant resource allocation to AMF may vary strongly among species [ 68 ]. The ecological importance of this interaction can be broadly appreciated; symmetric benefit transfer between plant host and AMF (positive feedback) may cause a decline in species diversity [ 69 ], while asymmetric benefit (negative feedback) may contribute to the coexistence of competing plant species [ 70 ]. Here the resulting dynamic may contribute to plant species coexistence. The dominant plant species are predicted to support growth and survival of subdominant species by providing mycorrhizal inoculum during the waterlogged season. While positive interactions among plants have been reported in wetland ecosystems, the mechanisms are mainly explained as protection from abiotic stress [ 13 ]. Our surveys and experiments show a strong positive effect of P. australis on soil oxygen availability, the major physical factor limiting the development of AMF in wetlands [ 71 ], and a positive association between P. australis and the proportion of I. polycephala roots associated with AM fungi. Field experiments demonstrate that P. australis removal leads to large decreases in AMF colonization, confirming that P. australis facilitates the presence of AM fungi. We also showed that experimental removal (both by fungicide application and P. australis exclusion) of AM fungi leads to large reductions in I. polycephala biomass, while, in the benomyl application treatment, neighbor removal did not decrease plant biomass, showing that the primary mechanism by which P. australis augments I. polycephala plant growth is the facilitation of mycorrhizal association. Until recently, AMF were considered to be unimportant in wetland communities [ 41 ], but our results demonstrate their potential importance in driving plant interactions in a marshland of the Yangtze River. The fact that AM fungi influence neighbor interactions involving subdominant plant species suggests that AMF could be critical in maintaining host plant species richness in this marshland community. However, as only five species were evaluated, establishing the generality of these results requires further substantiation. Further research will also be required to explore the response of AM fungal communities to waterlogging and their feedback to plant interactions and plant community structure and to quantify the relative importance of AM fungi to abiotic factors (e.g., waterlogging) as a driver of community structure and species diversity in marshlands."
} | 3,862 |
22131644 | PMC3210360 | pmc | 5,998 | {
"abstract": "Respiration and photosynthesis are two important processes in microalgal growth that occur simultaneously in the light. To know the rates of both processes, at least one of them has to be measured. To be able to measure the rate of light respiration of Chlorella sorokiniana , the measurement of oxygen uptake must be fast, preferably in the order of minutes. We measured the immediate post-illumination respiratory O 2 uptake rate (OUR) in situ , using fiber-optic oxygen microsensors, and a small and simple extension of the cultivation system. This method enables rapid and frequent measurements without disturbing the cultivation and growth of the microalgae. Two batch experiments were performed with C. sorokiniana in a short light-path photobioreactor, and the OUR was measured at different time points. The net oxygen production rate (net OPR) was measured online. Adding the OUR and net OPR gives the gross oxygen production rate (gross OPR), which is a measure for the oxygen evolution by photosynthesis. The gross OPR was 35–40% higher than the net OPR for both experiments. The respiration rate is known to be related to the growth rate, and it is suggested that faster algal growth leads to a higher energy (ATP) requirement, and as such, respiratory activity increases. This hypothesis is supported by our results, as the specific OUR is highest in the beginning of the batch culture when the specific growth rate is highest. In addition, the specific OUR decreases toward the end of the experiments until it reaches a stable value of around 0.3 mmol O 2 h −1 g −1 . This value for the specific OUR is equal to the maintenance requirement of C. sorokiniana as determined in an independent study of (Zijffers et al. 2010 (in press)). This suggests that respiration could fulfill the maintenance requirements of the microalgal cells.",
"introduction": "Introduction Respiration and photosynthesis are two important processes in microalgal growth that occur simultaneously in the light. These two processes and their relationship are extensively studied in plant science as reviewed by several authors (Badger et al. 1998 ; Geider and Osborne 1989 ; Graham 1980 ; Hoefnagel et al. 1998 ; Hunt 2003 ; Raghavendra et al. 1994 ; Turpin et al. 1988 ) because the balance between them determines, to a large extent, the growth and yield of most plants (Hunt 2003 ). In illuminated microalgal cells, three processes in which oxygen is involved occur simultaneously. These processes are schematically shown in Fig. 1 . The first process is photosynthesis in which oxygen is released, and ATP and NADPH are produced to be able to fix CO 2 into glyceraldehyde 3-phosphate (GAP). This can then be converted into biomass building blocks. The second process is respiration. This process mainly takes place in the mitochondria where NADH is oxidized to generate extra energy in the form of ATP to support biomass formation and maintenance processes. In this process, oxygen is consumed (Geider and Osborne 1989 ; Graham 1980 ; Hoefnagel et al. 1998 ; Turpin et al. 1988 ). The third process that can occur in the light is photorespiration. The oxygenase activity of Rubisco can also fix oxygen instead of carbon dioxide, forming glycolate. Glycolate is converted into glyceraldehyde 3-phosphate, so it can be re-used in biosynthesis. During this process, CO 2 and ammonia are lost, which need to re-fixed. This demands substantial energy in the form of ATP and NADPH (Tural and Moroney 2005 ). The process of photorespiration occurs at high extracellular oxygen concentrations or at low carbon dioxide concentrations and can be neglected when this is not the case (Peltier and Thibault 1985 ). Photorespiration is a complex process of which the reactions are divided over several cell organelles. This is reviewed extensively by Bauwe et al. ( 2010 ), Foyer et al. ( 2009 ), Maurino and Peterhansel ( 2010 ), Ogren ( 1984 ), and Wingler et al. ( 2000 ) among others. Figure 1 shows a simplified overview of photorespiration.\n Fig. 1 Simplified overview of an algal cell in the light, showing the processes in which oxygen and energy in the form of ATP are either consumed or produced. In the chloroplast, light is fixed ( 1 ), yielding O 2 , NADPH, and ATP. These are needed for the fixation of carbon dioxide by Rubisco into glyceraldehyde 3-phosphate (GAP) ( 2 ). GAP can be regenerated into Ribulose 1,5-bisphosphate using ATP ( 3 ) or can be transported to the cytosol ( 4 ) to be converted into building blocks for biomass ( 5 ). The oxygenase activity of Rubisco can also fix O 2 , forming glycolate ( 6 ). This process is called photorespiration. Energy is consumed to convert glycolate into 3-phosphoglycerate (3-PGA) and eventually into GAP ( 7 ), so it can enter the central carbon metabolism. During this process, CO 2 and ammonium ( \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ {\\hbox{N}}{{\\hbox{H}}_4}^{ + } $$\\end{document} ) are ‘lost’ and need to be re-fixed elsewhere in the metabolism, costing more energy. Energy in the form of ATP is yielded through the glycolysis and TCA cycle ( 8 ). Electrons are carried via NADH and FADH 2 to the electron transport chain located in the membrane of the mitochondria ( 9 ), yielding more ATP by taking up O 2 \n \n To understand the energy metabolism of algal cells and with that the conversion of light energy into biomass, insight into the rates of these three processes is necessary. In this paper, we work under conditions where photorespiration can be neglected. In this situation, the net oxygen exchange rate, which can be directly measured, is the sum of the oxygen production by photosynthesis and oxygen consumption through respiration in the mitochondria. To know the rates of these two processes, one of these has to be measured. In this paper, we estimate the light respiration rate by measuring the post-illumination oxygen uptake. The rate of post-illumination O 2 uptake has been shown to provide a good measure for respiratory O 2 uptake in the light (Grande et al. 1989 ; Weger et al. 1989 ; Xue et al. 1996 ). In Chlorella pyrenoidosa , respiration rates decreased from an initially high rate immediately after transfer to darkness to a much lower rate after 12–24 h in darkness (Geider and Osborne 1989 ). Bate et al. ( 1988 ) found a decline of respiration to the basal rate of steady-state dark respiration within an hour upon transfer to darkness for Dunaliella tertiolecta . This suggests that to be able to measure the rate of light respiration of C. sorokiniana , the measurement of post-illumination oxygen uptake must be performed immediately upon transfer to darkness. In addition, at higher biomass concentrations, the oxygen uptake rate will be high, and therefore, the oxygen concentration will decrease to zero in the order of a few minutes. Therefore, an oxygen probe with a short response time is needed. Widely used methods to determine respiratory O 2 uptake in the light are gas analysis, mass spectrometry using oxygen isotopes and Clark-type oxygen electrodes. The advantages and disadvantages of these methods for O 2 measurements are reviewed by Hunt ( 2003 ), Suresh et al. ( 2009 ), and Millan-Almaraz et al. ( 2009 ). The main drawbacks of these methods are the time scale in which measurements are possible, and the fact that for some of the methods, the algae need to be transferred from the cultivation vessel to a measurement chamber. This can cause changes in growth rate and thus in respiration rate. Fast and in situ measurements of oxygen uptake are preferred and these can be done using luminescence-based O 2 sensors. These fiber-optic sensors offer advantages over electrochemical devices, such as lack of oxygen consumption, insensitivity to interfering agents, and, most important, a faster response time (López-Gejo et al. 2009 ). In addition, Tyystjarvi et al. ( 1998 ) found the same oxygen uptake data with fiber-optic sensors as with leaf disk O 2 electrodes, indicating that measuring OUR with fiber-optic sensors is a reliable method. This paper describes a new method to measure the rate of respiration of Chlorella sorokiniana in the light, in situ inside a short light-path (SLP) photobioreactor during cultivation, by means of a simple extension of the cultivation system. This is done by measuring the immediate post-illumination O 2 uptake using two types of commercially available fiber-optic oxygen microsensors. This method enables rapid and frequent measurements without disturbing the cultivation and growth of the microalgae. In the photobioreactor set-up used, the net oxygen production rate (OPR) is measured online using a gas analysis system. This net OPR represents the oxygen that is produced as a sum of all processes in the cell that either produce or consume oxygen. By measuring the oxygen uptake rate (OUR) by respiration and adding the amount of consumed oxygen to the amount of net produced oxygen, the gross OPR can be calculated giving the total rate of photosynthesis. Consequently, the method described in this paper gives insight into the different processes in which oxygen is involved in the light inside a microalgal cell, and more specifically, it gives insight into the energy requirements for biomass formation and maintenance.",
"discussion": "Discussion To be able to gain insight in microalgal respiration during photosynthesis in the light, we measured the oxygen uptake of the algae immediately upon transfer into darkness during different time points in the batch experiments. The post-illumination oxygen uptake rates were thus measured at different biomass concentrations and therefore at different light supply rates. Immediately upon transfer to darkness, photosynthesis stopped, and the O 2 present in the dark tube was taken up by the algae within minutes. Both optical microsensors were able to measure this uptake since the response time of both sensors was in the order of seconds. The measured decrease of oxygen is shown in Fig. 5 . This measurement was very similar to all other measurements we performed. On each measurement output, we performed linear regression, and we always found a R \n 2 of 0.95 or higher and a P value well below 0.05, indicating that the decrease of O 2 was linear. From these measurements, a slope could be calculated, indicating the volumetric OUR in mmol h −1 kg −1 . It is also interesting to note that during this measurement, no change in the slope could be detected. On the timescale of these measurements, the oxygen uptake rate did not decrease toward the lower respiration rate of dark adapted algae. This lower dark rate would be reached on a timescale of hours according to the literature (Bate et al. 1988 ; Geider and Osborne 1989 ), and with this fast measurement, we were not able to see this lower respiration rate of dark adapted algae. Therefore, the measured OUR must have been predominantly caused by mitochondrial (light) respiration. Another process that could cause O 2 uptake in the light is photorespiration, but we estimated that photorespiration only could take place at a low rate. Ogren ( 1984 ) described an equation to calculate the relative rate of photosynthesis versus photorespiration v \n c / v \n o for isolated Rubisco (Eq. 11 ). It is important to note that this equation was derived for free Rubisco enzymes and that several transport processes play a role in the functioning of Rubisco in whole cells. Currently, it is not possible to calculate the intracellular CO 2 and O 2 concentration at the site of Rubisco, and the selectivity of the free enzyme is the only way to estimate whether photorespiration takes place at the concentrations of O 2 and CO 2 present in the cultivation medium. The specificity factor S \n rel was determined experimentally to be 61 for Chlamydomonas reinhardtii (Ogren 1984 ).\n 11 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\frac{{{v_{\\rm{c}}}}}{{{v_{\\rm{o}}}}} = {S_{\\rm{rel}}}\\frac{{\\left[ {{\\text{C}}{{\\text{O}}_{{2}}}} \\right]}}{{\\left[ {{{\\hbox{O}}_{{2}}}} \\right]}} $$\\end{document} \n At the point of maximum productivity in experiment II, which was higher than for experiment I, the dissolved oxygen reached 122% air saturation corresponding to an oxygen concentration in the medium of 260 μmol L −1 . At this point, the concentration dissolved CO 2 was 102 μmol L −1 (corresponding to 1.26% v / v CO 2 in the gas phase, i.e., the logarithmic average of the ingoing and outgoing CO 2 volume fractions) as calculated by the method described by Royce and Thornhill ( 1991 ). This method is based on the measured OPR and dissolved oxygen concentration to calculate the mass transfer coefficient for O 2 and subsequently CO 2 , followed by the calculation of the dissolved carbon dioxide concentration. The ratio of photosynthesis and photorespiration v \n c / v \n o then becomes 23.93. This means that the rate of photorespiration is indeed very low, 4% of photosynthesis, at that point. During the rest of the experiment, the ratio of CO 2 to O 2 increased again so the rate of photorespiration will have been <4% of gross photosynthesis. This is much smaller than the difference between net and gross OPR, and this difference therefore must have been predominantly caused by mitochondrial respiration. Photorespiration could lead to the accumulation of photorespiratory intermediates, which could still be converted in the photorespiratory pathway upon transfer of the cells to darkness and lead to products that can be respired. This post-illumination effect could hypothetically lead to enhanced respiration in the first minutes after transfer to darkness. However, the slope of the respiration measurement (i.e., the rate of decrease of O 2 concentration, Fig. 5 ) did not decrease, indicating that this effect was negligible, which further supports our conclusion that photorespiration as a whole was negligible under the applied cultivation conditions. To show the total oxygen evolution by photosynthesis (the gross OPR), the online measured net OPR and the OUR were added up. This is shown in Fig. 6 . The graphs for both experiments generally show the same pattern, although the values for experiment II are slightly higher. This could be due to the use of different microsensors. It is known that the respiration rate is related to the growth rate (Falkowski et al. 1985 ). Therefore, when the algae are growing faster, more energy for growth is needed, and the OUR will be higher and, consequently, the gross OPR will be higher too as can be seen from our results. The OUR increases with increasing biomass, and when the PQ (as shown in Fig. 4 ) stabilizes, the OUR values also become relatively constant. Near the end of both experiments, the OUR starts to increase. When looking at the rates of O 2 uptake where the PQ is constant, the gross OPR was 35–40% higher than the net OPR for both experiments. This is in agreement with the 33% found by Weger et al. ( 1989 ) for Thalassiosira as measured by mass spectrometry, and the 17–43% found for cyanobacterial biofilms as measured with a Clark-type microsensor (Kühl et al. 1996 ). Bate et al. ( 1988 ) found a difference of 15.6% for the green alga D. tertiolecta . Compared to specific studies using C. sorokiniana , our values are high. In two studies, Vona et al. determined the difference between gross OPR and net OPR to be 5% for C. sorokiniana (Vona et al. 2004 , 1999 ). However, they used different culture conditions, and more importantly, they used a different, ex situ , method to measure the O 2 uptake rate. In the aforementioned study, the respiratory O 2 uptake was measured by transferring the cells to a biological oxygen meter (BOM) equipped with an oxygen electrode. Transferring the cells from the cultivation vessel to a BOM might result in a lower growth rate and a lower O 2 uptake of the cells. Similar to a previous study (Kliphuis et al. 2010 ), we observed an optimum OPR and CUR at a biomass concentration of around 2.3 g L −1 for both batch experiments with C. sorokiniana (Fig. 4a ). This point is the moment where the biomass yield on light energy ( Y \n x , E (obs) ) reaches its maximum value, 0.82 g mol −1 for experiment I and 0.87 g mol −1 for experiment II. At first, more light was available per cell. Because of this the algae could fix more CO 2 , produce more O 2 and grow faster. While the biomass concentration in the photobioreactor keeps increasing, as can be seen in Fig. 4b , the available light per cell decreases due to mutual shading, and the OPR and CUR reach an optimum. After this point, the OPR and CUR decrease again and keep on doing so until the end of the batch experiment. This decrease in net OPR and CUR after having reached the optimum can now be related to the specific OUR, which is shown in Fig. 7 . The specific OUR is highest at the optimum of the net OPR and decreases together with the net OPR until it reaches a stable value toward the end of the experiments. This trend can be explained by taking into account that part of the respiratory activity is coupled to growth, as discussed before, but that another part is related to the maintenance requirements of the microalgae. Maintenance in this context is defined as energy consumption for purposes other than growth (Pirt 1965 ). When there is sufficient light available per cell, the algae grow fast and need energy (ATP) to support growth. This energy is supplied by respiration of a part of the carbohydrates produced in photosynthesis, and thus, the specific OUR will be high. A portion of the available light per cell, on the other hand, is needed to generate energy for maintenance, possibly also via respiration of carbohydrates produced in photosynthesis. Thus, after having reached the optimum productivity, the cell density is such that all light is absorbed in the system. When the cell density increases further, the energy extracted from this light (via photosynthesis and respiration) will decrease from a maximal value during the optimum, consisting of a large growth-associated fraction and a smaller but constant maintenance-associated fraction, to a low value, composed of predominantly the constant maintenance-associated fraction. This analysis is supported by the maintenance requirement of C. sorokiniana , which was determined in an independent study of Zijffers et al. ( 2010 ). The specific OUR decreases toward the end of the experiments until it reaches a stable value of around 0.3 mmol O 2 h −1 g −1 . This low value for the specific OUR is in the same order as the value for maintenance requirement determined experimentally by Zijffers et al. ( 2010 ). Zijffers et al. ( 2010 ) determined a maintenance constant of 6.8 mmol photons g −1 h −1 for C. sorokiniana based on the maintenance/growth model by Pirt ( 1986 ). The corresponding yield Y \n x , E was 0.75 g mol photons −1 for growth on urea. The Y \n xE max for photoautotrophic growth on urea is estimated to be 1.8 g mol −1 . The maintenance constant therefore needs to be corrected for this growth inefficiency by multiplying with a factor 0.75/1.8, yielding a corrected maintenance constant of 2.84 mmol photons g −1 h −1 . In the light reaction of photosynthesis, eight photons are used to produce 1 mol O 2 . The formed ATP is used to fix 1 mol CO 2 into 1 C-mol biomass. When this biomass is respired again, 1 mol O 2 is taken up. When we assume that all O 2 uptake is due to maintenance, 1 mol O 2 is taken up per eight photons, giving 2.84 × 1/8 = 0.36 mmol O 2 g −1 h −1 . This value represents the OUR for maintenance only and corresponds well with the value found in our experiments. In short, Zijffers et al. determined the maintenance light requirements of C. sorokiniana based on the corresponding light use. In this study, we measured the specific oxygen consumption rate, which converged to a low and constant value at the end of the batch cultivation. Recalculating the data from Zijffers et al. to a specific respiratory oxygen consumption shows that both values are almost the same. This shows that respiration could fulfill the maintenance requirements of the microalgal cells. To conclude, the method described in this paper for measuring the respiration rates of microalgae proved to be a good technique for determining the oxygen uptake in situ during cultivation in a photobioreactor. Only a small and simple extension of the system was necessary to be able to measure respiration rates. This method enables rapid and frequent measurements without disturbing the cultivation and growth of the microalgae. The rate of oxygen uptake in the light gives insight in the gross oxygen evolution by photosynthesis, which is 30–45% higher than net oxygen evolution rate. Respiration rates in the light are very high, and consequently, photosynthesis rates are very high to produce sugars, which can be respired to produce extra ATP for growth. Measuring respiration rates during batch cultivation showed the relationship between growth and respiration as an energy supporting mechanism. It also provided strong evidence that respiration could fulfill the maintenance requirement of the microalgal cells. \n Nomenclature η Radius ratio of the photobioreactor (-) μ t Specific growth rate at time t (h -1 ) A pbr Illuminated photobioreactor area (m 2 ) CUR Carbon dioxide uptake rate (mmol h -1 ) CUR cum Cumulative carbon dioxide uptake rate (mol) C x Biomass present in the photobioreactor as measured with dry weight measurement (g) C x,tot Total biomass present in the photobioreactor, including biofilm, calculated from CO 2 uptake (g) K m affinity constant for carbon dioxide (μmol L -1 ) M biomass Molar mass of dry biomass (g mol -1 ) m E,x Maintenance coefficient (mol g -1 h -1 ) n gas,in Total molar gas flow going into the reactor (mmol h -1 ) n gas,out Total molar gas flow going out of the reactor corrected for moisture content (mmol h -1 ) OPR Oxygen production rate (mmol h -1 ) OPR gross Gross oxygen production rate (mmol h -1 ) OUR Oxygen uptake rate (mmol h -1 ) OUR corr Oxygen uptake rate corrected for biofilm in the photobioreactor (mmol h -1 ) OUR spec Oxygen uptake rate per unit biomass (mmol h -1 g -1 ) PAR Photosynthetic active radiation, all photons between 400 and 700 nm PF in Photon flux on the surface of the photobioreactor (mmol h -1 ) PFD in Photon flux density on the surface of the photobioreactor (μmol m -2 s -1 ) PQ Photosynthetic quotient (-) r E,x Light supply rate (μmol g -1 s -1 ) r i Radius of inner (rotating) cylinder (m) r o Radius of outer (stationary) cylinder (m) V pbr Photobioreactor volume (kg) x CO2,db Molar fraction of CO 2 in dry baseline (-) x CO2,exp Molar fraction of CO 2 in experimental gas data (-) x CO2,wb Molar fraction of CO 2 in wet baseline (-) x O2,exp Molar fraction of O 2 in experimental gas data (-) x O2,wb Molar fraction of O 2 in dry baseline (-) Y x,E Biomass yield on light energy (g mol -1 ) Y xEmax Maximal yield of biomass on light energy (g mol -1 ) Y x,E(obs) Observed biomass yield on light energy (g mol -1 )"
} | 5,902 |
38500621 | PMC10945513 | pmc | 5,999 | {
"abstract": "Microbial fuel cells (MFCs) represent simple devices that harness the metabolic activities of microorganisms to produce electrical energy from diverse sources such as organic waste and sustainable biomass. Because of their unique advantage to generate sustainable energy, through the employment of biodegradable and repurposed waste materials, the development of MFCs has garnered considerable interest. Critical elements are typically the electrodes and separator. This mini-review article presents a critical assessment of nanofiber technology used as electrodes and separators in MFCs to enhance energy generation. In particular, the review highlights the application of nanofiber webs in each part of MFCs including anodes, cathodes, and membranes and their influence on energy generation. The role of nanofiber technology in this regard is then analysed in detail, focusing on improved electron transfer rate, enhanced biofilm formation, and enhanced durability and stability. In addition, the challenges and opportunities associated with integrating nanofibers into MFCs are discussed, along with suggestions for future research in this field. Significant developments in MFCs over the past decade have led to a several-fold increase in achievable power density, yet further improvements in performance and the exploration of cost-effective materials remain promising areas for further advancement. This review demonstrates the great promise of nanofiber-based electrodes and separators in future applications of MFCs.",
"conclusion": "5. Conclusions This article described the significance of nanofiber technology in revolutionizing MFCs as a sustainable energy source. The proper and efficient selection of the material from which MFCs are constructed is a crucial element in the effort to produce high-performance MFCs. Nanofiber technology has been shown to enhance the performance of MFCs through improved electron transfer rate, enhanced biofilm formation and microbial activity, and increased durability and stability. Nanofiber-based anodes, cathodes, and membranes have been investigated in MFCs, with promising results. However, the incorporation of nanofiber technology into MFCs poses various challenges encompassing cost-effectiveness, biocompatibility, and long-term stability. Future research should focus on the optimizing the properties of nanofiber and the exploration of their potential applications not only in MFCs but also in other energy-related domains. Utilizing MFCs as a viable source of sustainable energy presents an opportunity to reduce reliance on non-renewable resources and address the impacts of climate change. Additionally, the potential of nanofiber technology to improve MFC performance and expand their applications holds great promise in advancing a future driven by sustainable energy solutions.",
"introduction": "1. Introduction The utilization of biomass, particularly organic waste, is considered an environmentally friendly and sustainable approach to energy production, making it a valuable alternative source of renewable energy. 1 Microbial fuel cells (MFCs) have gained increasing attention as promising bio-electrochemical systems that can convert chemical energy stored in organic compounds, such as acetate, sugars, nitrate, and ethanol, 2,3 into electricity through the metabolic activity of microorganisms. 4,5 MFCs offer numerous advantages over conventional fuel cells, including negating the need for expensive or exotic catalysts, such as platinum, and generating electricity from renewable sources of organic matter, including waste streams. 6 Furthermore, MFCs have the potential to remove pollutants via the microbial metabolism thereby finding application in wastewater treatment, bioenergy generation, and biosensors. 5,7,8 By definition, the MFC is a device that converts the energy from organic compounds into electrical energy through the metabolic processes of microorganisms. 9–12 The operation of MFCs is based on the transfer of electrons from the anode electrode to the cathode. This is achieved by electrochemically active bacteria, which oxidize organic matter in the anode compartment, releasing electrons and cations, eqn (1) . The electrons flow through the external circuit to the cathode, where they combine with an oxidant to produce water ( eqn (2) ). 7,13,14 Meanwhile, the protons migrate through the membrane to the cathode compartment, where they combine with the electrons and an oxidant ( e.g. , O 2 ) to complete the reaction, as per the given chemical eqn (2) . 13 1 C 2 H 4 O 2 + 2H 2 O → 2CO 2 + 8e − + 8H + 2 2O 2 + 8H + + 8e − → 4H 2 O ( E 0 = 1.23 V) In a conventional MFC, two half-cells – an anode and a cathode, are separated by an ion exchange membrane, as depicted in Fig. 1 . The process of electricity generation in the MFC is sustained through a continuous consumption of an oxidising agent, e.g. , oxygen, as indicated by eqn (1) and (2) . The cathode compartment can work with either aqueous or atmospheric oxygen. 15 Due to its high redox potential, oxygen is considered to be a suitable electron acceptor for the cathode in MFCs. The interest in microbial fuel cells has been consistently increasing over the last two decades. 16 Fig. 1 The schematic and fundamental principles of a conventional microbial fuel cell (MFC). Despite these promising features and significant interest, the performance of MFCs is currently characterized by lower power density, when compared with chemical fuel cells, whose rates of reaction are naturally higher than biological processes; this has however driven the need for innovation to enhance performance. As with every real system, MFCs produce energy output that is lower than their theoretical maximum due to different electrochemical losses. The losses are due to resistance in materials, separator material, and electrolytes, leading to the lower power production of MFCs compared to their potential. According to Torres et al. , 17 the primary obstacle for maximizing power output in microbial fuel cells (MFCs) is the reactor design, which must integrate anodes with a high surface area, low ohmic resistances, and minimal cathode potential losses. Developments in electrode and membrane materials are focused on enhancing MFC performance by seeking novel materials with improved capabilities. 18–21 In recent years, nanofibers in MFCs have emerged as a possible pathway to enhancing performance. Carbon nanofibers (CNFs) are widely utilised as MFC electrodes due to their unique network structure and exceptional structural stability. The main challenges for MFC systems are cost reduction and productivity enhancement. Using nanofibers offers a viable option to tackle the main issues of reducing costs and increasing productivity in microbial fuel cell (MFC) systems. 22,23 Nanofibers can be produced economically employing efficient methods and resources, leading to decreased production expenses. The customisable features enhance the optimisation of electrode and membrane materials, hence enhancing the performance and lifespan of MFC systems. Due to their small size, highly porous structure, tight pore size, and high specific surface area, nanofiber webs are ideal for integration into MFCs. 24,25 Such properties offer several advantages in MFCs, including enhanced bacterial adhesion, mass transfer, and electron transfer efficiency. 26,27 Fig. 2 illustrates the superior power generation advantages of CNFs anode compared to commercial carbon felt. For instance, incorporating nanofibers into the anode can promote microbial adhesion and increase surface area, resulting in faster electron transfer rates and higher power production. 28,29 Tao et al. 30 used a hierarchically structured textile polypyrrole/poly(vinyl alcohol-co-polyethylene) nanofibers/poly(ethylene terephthalate) (referred to PPy/NFs/PET) as an anode of MFC. The results showed the high surface roughness, porous and three-dimensional interconnecting conductive scaffold improved the colonization of Escherichia coli and electron transfer to the anode. The maximum power and current densities were 2420 mW m −2 and 5500 mA m −2 , which is approximately 17 times higher compared to anode prepared without a nanofiber layer (144 mW m −2 ). It is clearly shown that the nanofiber effect on the colonization of bacteria is non-negligible. Fig. 2 Composite anode of electrospun carbon nanofibers and hybrid carbon nanotubes facilitates microbial attachment, electron transfer, and exhibits superior conductivity and biocompatibility compared to commercial carbon felt (this figure has been reproduced from ref. 26 with permission from Elsevier publisher, copyright 2024). Integrating nanofibers in MFCs has demonstrated potential benefits for improving power density, current output, and durability of these cells. Nanofibers can be used as an anode material to facilitate electron transfer from bacteria to the electrode surface, a cathode material to enhance oxygen reduction, or a membrane material to separate the anode and cathode compartments. However, more research is needed to optimize the fabrication and integration of nanofibers in MFCs and to understand their long-term stability and performance under different operating conditions. In the literature, CNFs have been widely studied in MFCs due to their excellent electrical conductivity and biocompatibility. One such study, 31 employed activated electrospun carbon nanofibers (ACNFs) in an MFC as an alternative cathode catalyst to platinum (Pt) and conducted a performance comparison with plain carbon paper. It was found that chemically ACNFs showed better catalytic activity than that of the physically activated one with 78% more power generation. Chemically ACNFs with 8 M KOH generated oxygen reduction reaction (ORR) performance levels that contributed to 3.17 times more power than that of the carbon paper, 1.78 and 1.16 times more power generation than that of the physically activated ACNFs and the chemically activated ACNFs with 4 M KOH, respectively. Karra et al. 32 utilized ACNFs as the anode material to stimulate bacterial biofilm growth, and improve MFC performance. The analysis of biofilm adhesion, both qualitatively and quantitatively, indicated that ACNFs outperformed other commonly used carbon anodes. The power density of the ACNFs was 1.13 and 3.18 times higher than that from granular activated carbon and carbon cloth anodes, respectively. Metal doped carbon nanofibers (MDCNFs) have also been explored for their use in MFCs due to their high electrical conductivity and catalytic activity. Bosch-Jimenez et al. 33 have successfully prepared CNFs doped with metals such as Co, Ni or Fe which increased surface areas up to 573 m 2 g −1 . Adding metals increased mesoporosity and catalytic activity of cathode material. Manickam et al. 29 used activated carbon nanofiber anodes in MFC. The preliminary tests in a single chamber MFC demonstrated a 10% increase in current densities to ∼2715 A m −3 compared to the highest maximum obtained so far. The bio-electrochemical performance of activated carbon nanofiber anodes was compared to commonly-used anodes like carbon cloth and granular activated carbon, and this anode architecture is expected to help overcome low power density issues that have limited the widespread adoption of MFCs. Polymer nanofibers have been investigated for their use in MFCs due to their high surface area and flexibility. Polymeric polyvinylidene fluoride (PVDF)/Nafion composite membranes are good candidates as proton exchange membranes in MFCs due to their porosity, high specific surface area, tight pore size, chemical resistance, good electrical insulation, good thermal properties and its biocompatibility 34,35 as shown in Fig. 2 . When combining carbon nanofibers (CNFs) with Nafion 117, a commonly utilized membrane in Microbial Fuel Cells (MFCs), can alter membrane roughness, pore size, and porosity, consequently enhancing the power generated by the MFCs. 36 The reduction in pore size and roughness of these nanocomposite membranes leads to the blockage of oxygen transfer from the cathode to the anode and impedes the migration of bacteria and other components from the anode to the cathode. Consequently, higher power production can be achieved. Chae et al. 37 developed a sulfonated polyether ether ketone (SPEEK)-based composite proton exchange membrane reinforced with polyimide nanofibers for use in microbial electrolysis cells. The addition of the nanofiber layer not only enhances the dimensional stability of the SPEEK membrane but also improves its affinity for protons, all while reducing costs. Additionally, the composite membrane demonstrated superior hydrogen efficiency (electron to hydrogen) of 86.4 ± 14.7%, compared to 77.2 ± 10.3% observed with Nafion membranes. In addition to the potential improvement of MFC performance, nanofiber technology can also contribute to the sustainability of MFCs by utilizing renewable feedstocks in nanofiber production. Nanofibers can be fabricated from various materials, such as carbon, metal, polymer, and ceramic, using different fabrication techniques, including electrospinning, 38,39 melt spinning, 40 force spinning, 41 chemical vapour deposition, 42 and template synthesis. 43 The most common nanofiber production process is electrospinning due to several benefits compared to conventional techniques for producing nanofibers, including flexibility in choosing materials, ability in controlling fiber size and structure, and capacity for large-scale production. Electrospinning is a method that can produce continuous and uniform nanofibers from various polymers and composite materials, making it a popular choice for applications in various applications, including fuel cells."
} | 3,456 |
35521541 | PMC9062269 | pmc | 6,000 | {
"abstract": "Summary Liquid phase leakage, intrinsic rigidity, and easy brittle failure are the longstanding bottlenecks of phase change materials (PCMs) for thermal energy storage, which seriously hinder their widespread applications in advanced energy-efficient systems. Emerging flexible composite PCMs that are capable of enduring certain deformation and guaranteeing superior mutual contact with integrated devices are considered as a cutting-edge effective solution. Flexible PCMs-based thermal regulation technology can reallocate thermal energy and regulate the temperature within an optimal range. Currently, tireless efforts are devoted to the development of versatile flexible PCMs-based thermal regulation devices, and a big step forward has been taken. Herein, we systematically outline fabrication techniques, flexibility evaluation strategies, advanced functions and advances of flexible composite PCMs. Furthermore, existing challenges and future perspectives are provided in terms of flexible PCMs-based thermal regulation techniques. This insightful review aims to provide an in-depth understanding and constructive guidance of engineering advanced flexible multifunctional PCMs.",
"conclusion": "Conclusions and future prospects Multifunctional PCMs have been widely explored for advanced latent heat storage systems. Extensive utilization of PCMs in thermal regulation can efficiently heat or cool human body, building and electric battery etc. Nevertheless, liquid leakage, intrinsic rigidity and easy brittle failure are the long-standing bottlenecks of conventional PCMs, which seriously hinder their widespread applications. Facing low-carbon and green strategic demands, therefore, great efforts have been invested in flexible thermal regulation systems based on shape-stabilized composite PCMs, and advanced applications have been explored to satisfy diverse thermal regulation requirements. Importantly, flexible composite PCMs can effectively overcome the long-standing bottlenecks mentioned above, and can guarantee superior mutual contact with integrated devices, thereby providing great application potential. In this review, we provide a comprehensive overview of preparation strategies, flexibility enhancement, flexible mechanisms, thermal performance and the state-of-the-art applications of flexible PCMs-based thermal regulation systems from the perspective of material chemistry. Compared with conventional composite PCMs, flexible shape-stabilized composite PCMs exhibit remarkable advantages, including excellent flexibility, lightweight, intelligence, and wearability. Based on the flexibility mechanisms, we focused on two preparation strategies: physically encapsulating PCMs into flexible supporting materials, and chemically grafting PCMs onto the supporting materials. In regard to flexible composite PCMs, we creatively divided them into 1D (cellulose, CNTs and other 1D materials), 2D (graphene, BN and MXene) and 3D (EG, sponge and other 3D materials) flexible composite PCMs according to the structural dimensionality. Benefiting from significant thermal performances, shape evolution and adaptability, flexible composite PCMs exhibit remarkable achievements in air-conditioning, thermal regulation of human body, electronic devices and batteries, thermal therapy, shape memory and solar/electro-thermal energy conversion. Although numerous important advancements have been made in flexible shape-stabilized composite PCMs for thermal regulation systems, more efforts and challenges are still required to further investigate and conquer in the future. 1) Currently, there is a serious conflict between the flexibility and thermal storage capacity of composite PCMs. Further research should focus on how to balance the flexibility and thermal storage capacity of composite PCMs. 2) High-performance flexible composite PCMs with higher thermal conductivity should be further developed for better thermal regulation. 3) The development of new flexible wearable and self-repairing energy storage devices based on PCMs integrated with other advanced functions is a promising research direction. 4) The flexibility mechanisms of flexible shape-stabilized composite PCMs need more systematic explanations for better practical applications. 5) To realize the practical application of flexible PCMs in complex and harsh conditions, stable flexibility and durability should be guaranteed under different conditions. Further researches should focus on the stability and durability of flexible PCMs. 6) Specific evaluation criteria, assessment index, and test standards of flexible PCMs (such as bending and twisting angle, tensile length, fatigue test, etc.) should be established in further research. In addition, flexible characterization techniques need further development, such as extension test, pre-stretching and nanoindentation techniques. 7) Exploring low-cost, large-scale and refined production technology of flexible PCMs is a very valuable research direction, although the road is very long and bumpy. 8) The potential and emerging applications of flexible PCMs should be further developed such as thermoelectric conversion, nanogenerator, electromagnetic shielding, etc.",
"introduction": "Introduction Thermal regulation technology is an efficient approach to reallocate thermal energy for both heating and cooling of the human body, buildings and electric batteries and so on ( Chen et al., 2020d ; Moore and Shi, 2014 ; Wang et al., 2020b ; Weinstein et al., 2018 ; Zhang et al., 2020 ). Although various thermal regulation technologies have been developed in the past years, they suffer from some intrinsic drawbacks ( Hsu et al., 2016 ; Huang et al., 2016 ; Lin et al., 2021a ; Tsai et al., 2020 ; Westwood et al., 2021 ). For example, integrating active air/liquid-cooling technology into rechargeable batteries can prevent overheating and thermal runaway at high temperatures, but they are usually bulky, heavy, complicated in structure, and high in power consumption. Moreover, it is necessary to heat the batteries at a low ambient temperature to achieve efficient working performance and safe operation of batteries ( Kant et al., 2019 ; Lin et al., 2021a ; Liu et al., 2019 ; Lizana et al., 2019 ; Yang et al., 2019 ; Zhu et al., 2019 ). Similarly, the thermal management devices of the human body in hot or cold environments are also crucial to human health. Various personal body heating/cooling technologies, such as photo heating, electric heating, ice cooling, water cooling, and air cooling, have been developed ( Booten et al., 2021 ; Florindo et al., 2018 ; Hsu et al., 2017 ; Sipponen et al., 2020 ; Yang et al., 2017 ). However, these thermoregulation technologies usually restrict the mobility of the human body because of the heavy and complex wearable devices. Both the thermal regulation of human bodies and batteries can merely provide cooling or heating separately, but cooling and heating are usually simultaneously required under various conditions in many scenarios ( Chen et al., 2020f ; Ci et al., 2019 ; D'Alessandro et al., 2018 ; Waqas et al., 2018 ; Yan et al., 2021 ; Yang et al., 2021 ). In addition, thermal regulation needs to consider the diverse requirements of individuals, which can improve personal thermal comfort. It is worth noting that the traditional thermal regulation will not operate quickly once the external energy is removed. Therefore, it is of great importance to integrate heating/cooling thermal regulation into a small-scale system to adapt to a wider temperature range and extend the thermal conditioning time even in the case of removing external energy. In response to the above critical issue, recently, an efficient solution based on solid-liquid phase change materials (PCMs) has been proposed which can regulate the temperature passively by utilizing large phase transition enthalpy of PCMs during the reversible crystalline-amorphous process ( Hyun et al., 2014 ; Liu et al., 2021a ; Velasco et al., 2021 ). Although PCMs can perform effective thermal regulation in various scenarios by absorbing and releasing thermal energy in the form of latent heat during the phase transition process, liquid PCMs are easy to flow, which restricts their widespread applications ( Amaral et al., 2017 ; Chen et al., 2020g ; Dai et al., 2021 ; Liu et al., 2021c ; Saffari et al., 2017 ). To solve the liquid leakage issue of PCMs, diverse strategies have been developed to fabricate shape-stabilized composite PCMs such as core-shell encapsulation (microcapsule, CNTs, etc.), interface encapsulation (graphene, MXene, etc.) ( Kumar et al., 2020 ; Shao et al., 2021a ), and porous encapsulation (MOFs, EG, etc.) ( Chen et al., 2020e , 2020h , 2021 ; Huang et al., 2020 ; Kashyap et al., 2019 ; Liu et al., 2021d ; Wang et al., 2021b ). Unfortunately, the shape-stabilized composite PCMs usually exhibit strong rigidity and poor flexibility, resulting in difficult installation, easy brittle failure and poor surface contact ( Abdelrazik et al., 2020 ; Ashraf et al., 2017 ; Graham et al., 2020 ; Liu et al., 2020 ). The high thermal contact resistance caused by these problems is not conducive to efficient thermal regulation. To develop flexible PCMs-based thermal management devices, liquid phase leakage and solid phase rigidity of conventional PCMs should be addressed. Therefore, the thermal regulation systems based on PCMs are developing in the direction of flexibility, lightweight, intelligence, and wearability, which puts forward higher requirements for the mechanical strength and flexibility of PCMs. Very recently, flexible engineering of PCMs technologies has attracted a lot of attention and offer a new avenue for significant improvement in the mechanical properties of PCMs while maintaining their energy storage capacity. High flexibility can guarantee that the thermal regulation materials contact the surface most fully under the condition of low installation pressure, and it is convenient to install and disassemble ( Alam et al., 2019 ; Mandal et al., 2019 ; Wu et al., 2019c , 2021a ). It is worth mentioning that mechanical flexibility and energy storage density are two contradictory evaluation factors for flexible composite PCMs. Flexible supporting materials are generally stretchable, twistable, foldable, and bendable and their flexible performances keep almost unchanged even though after suffering many deformation cycles. However, the flexibility of the supporting materials will be gradually destroyed with the increase of the content of PCMs. This is because PCMs tend to display a rigid state when solidified; the presence of a rigid component will restrict the flexible movement of supporting materials ( Lin et al., 2022 ; Petruo et al., 2021 ; Ren et al., 2022 ). Currently, the two main methods for developing flexible thermal regulation systems based on PCMs are encapsulating PCMs into flexible supporting materials, which is a physical approach based on capillarity or hydrogen bonding, and grafting PCMs onto the supporting materials, which is a chemical approach based on grafting reaction. It should be noted that the supporting materials are importantly required no matter which method ( Chen et al., 2018 ; He et al., 2019 ; Jing et al., 2019 ; Zayed et al., 2019 ) is adopted. Recent reviews mainly concentrate on the encapsulation strategies, thermal conductivity enhancement, and applications of PCMs in energy conversion and storage ( Aftab et al., 2018 ; Chandel and Agarwal, 2017 ; Chen et al., 2020g ; Cheng et al., 2021 ; Frigione et al., 2019 ; Li and Mu, 2019 ; Shchukina et al., 2018 ; Wu et al., 2021b ; Yuan et al., 2020 ), or focus on the preparations, properties and applications of flexible PCMs ( Shi et al., 2021 ). However, an insightful understanding of flexible engineering of advanced PCMs is still insufficient. From the perspective of material science, herein, we systematically outline a comprehensive review of flexible PCMs based on different dimensional flexible additives, including 1D flexible additives, such as cellulose( Wei et al., 2019 ) and carbon nanotubes (CNTs) ( Wang et al., 2021a ); 2D flexible additives, such as graphene and their derivatives( Li et al., 2018 ), boron nitride (BN) ( Wang et al., 2020a ) ( Yang et al., 2018 )and MXene( Gong et al., 2021 ); and 3D flexible additives, such as EG( Wu et al., 2020c ), foam( Chang et al., 2020 ) and other 3D flexible materials 157 ( Figure 1 ). Moreover, we highlight fabrication techniques, flexibility evaluation strategies, advanced applications, current developments and further perspectives for flexible composite PCMs. This insightful overview aims to provide constructive guidance for the future development of advanced flexible PCMs. Figure 1 Layout structure of this review Overview of using different dimensional supporting materials (1D, 2D, 3D) for preparation of flexible composite PCMs and advanced multifunctional applications: thermal management, wearable textiles, solar-thermal, electro-thermal, thermotherapy, and shape memory."
} | 3,275 |
36530289 | PMC9753497 | pmc | 6,001 | {
"abstract": "In this paper, catalyst-free room-temperature healing\nepoxy vitrimer-like\nmaterials (S-vitrimer) are introduced. The S-vitrimer can be healed\nat room temperature without any external stimuli such as solvent,\npressure, heat, and catalyst through an aromatic disulfide exchange\nreaction and a hydrogen bond because the glass transition temperature\nof the S-vitrimer is lower than room temperature. Self-healing materials\nare attracting widespread attention nowadays with their potential\nto increase the durability of the materials. However, there is still\nelevating need for research, considering the limitations of various\nself-healing methods. To the best of our knowledge, epoxy-based catalyst-free\nroom-temperature healing materials have not been investigated until\nnow, yet they are promising to make self-healing easier. Moreover,\nthe S-vitrimer showed higher healing efficiency when healed for a\nlonger time and at a higher temperature. Especially when healed at\nroom temperature for 96 h, the S-vitrimer presented an 80% healing\nefficiency. The S-vitrimer also showed an 80% healing efficiency when\nhealed at 60 °C for 48 h. To investigate the factors affecting\nself-healing behavior, three control experiments were carried out.\nControl experiments showed that the S-vitrimer is healed mainly due\nto a disulfide exchange reaction, but hydrogen bonds also contribute\nto self-healing behavior. Also, it was found that tightly packed segments\ncan hinder self-healing through control experiments.",
"conclusion": "4 Conclusions In this paper, epoxy-based\nself-healing materials (S-vitrimer)\nthat can be healed without any external stimuli were synthesized.\nTo the best knowledge of the authors, it is the first time that an\nepoxy network healable without any stimuli at room temperature is\nreported. The S-vitrimer was characterized by FTIR, DSC, DMA, and\nTGA. Also, various control experiments were conducted to describe\nthe working mechanism of the overall self-healing process in detail. This study explored that the S-vitrimer can be healed at even room\ntemperature with an aromatic disulfide exchange reaction and a hydrogen\nbond. When the S-vitrimer was left in contact for a longer time, a\nhigher healing efficiency was observed. In particular, the S-vitrimer\nrecovered about 80% of the pristine strength at room temperature at\n96 h and 60 °C in 48 h. Finally, three control experiments\nwere conducted to demonstrate\nthe parameters affecting the self-healing behavior. First, the C1\nexperiment without disulfide was performed, and a 6.4% healing efficiency\nwas achieved. Through C1, it was found that the disulfide exchange\nreaction played an essential role in self-healing behavior, but the\nimportance of a hydrogen bond is also pointed out in healing behavior.\nC2 experiments without a pendant group were conducted, and the sample\nrecovered 45% of its original strength after self-healing. The pendant\ngroup implements more hydrogen bonds to the S-vitrimer, which directly\naffects the self-healing behavior. Finally, C3 was synthesized with\na different hardener formed of para -substitution\nin which healing behavior is not observed. C3 results suggested that\na tightly packed structure prevents high healing efficiency. Optimization\nof such a system offers a new generation of epoxy-based composite\nmaterials with the capability of self-healing even at room temperature.\nIn the future, care can be taken to further enhance mechanical strength.",
"introduction": "1 Introduction Living organisms can be\nhealed autonomously after being wounded,\nand they can recover their functions even if damaged. Inspired by\nnature, many researchers attempt to devise such a property in industrial\nmaterials to increase the durability and safety of the component.\nSelf-healing materials are attracting enormous attention nowadays,\nincluding various industries such as aerospace, 1 , 2 automobile, 3 , 4 electronics, 5 − 7 and robotics. 8 − 10 Many self-healing materials\nare fabricated employing reversible\ncovalent bonds since they promise self-healing multiple times in the\nsame area, avoiding additional chemicals such as healing agents or\ncapsules. The Diels–Alder reaction, 11 , 12 boronic ester formation, 13 , 14 disulfide exchange\nreaction, 15 − 18 imine reaction, 19 , 20 urea exchange reaction, 21 , 22 and transesterification 23 − 25 are the reversible covalent bonds\npreferred to synthesize self-healing materials mostly. However, in\nmost cases, self-healing requires external stimuli such as light, 5 , 25 − 27 solvent, 28 , 29 heat, 23 , 30 − 33 or a catalyst. 34 , 35 In such cases, the self-healing\npolymers cannot respond immediately under fracture because the healing\nmechanism needs to be triggered by external stimuli. Similarly, the\nincorporation of a catalyst may cause shortcomings in many ways, including\nbut not limited to aging, stability, and toxicity. In this scenario,\nthe state-of-art is heading toward new industry-friendly\nconcepts to overcome the difficulties of applying external stimuli\neach time. Even though a few papers reported self-healable polymers\nat room temperatures without external stimuli or catalysts via a disulfide\nbond, these self-healable polymers mainly focused on thermoplastics\nand other amorphous polymers such as thermoplastic polyurethane (TPU). 17 , 18 However, TPUs are not preferred in structural components due to\nlimitations in their utility at high temperatures. In other words,\nTPUs cannot be used in the aerospace industry where resistance to\nhigh temperature and high structural performance are required. 36 On the other hand, epoxy having a network structure\npossesses high heat resistance, which motivates its utility in tires\nand seals such as packing and gaskets in aircraft or spacecraft. 37 Moreover, epoxy is more facile to fabricate\nthan TPU and more ecofriendly due to the solvent-free and catalyst-free\nsynthesis process. 38 Even though there\nwere a few studies about epoxy materials healable within reversible\ncovalent bonds, 34 , 39 − 41 these research\nstudies addressed healing with a stimulus as mentioned above, which\nrequired an additional effort. Zou et al. 39 studied self-healing epoxy coatings based on Diels–Alder\nreactions, including MXene flakes that can be activated by near-infrared\n(NIR) light. They observed that having MXene content allowed the epoxy\nto convert NIR light energy to heat quickly, enabling self-healing\nfor nearly around 15 min. However, in such concepts, a specific wavelength\nof light and high contents of flakes, capsules, powders, etc., to\nabsorb light efficiently might be required, affecting the mechanical\nproperties of the composite. Moreover, a self-healing mechanism based\non Diels–Alder reactions might lead to a sudden viscosity drop,\nrisking the dimensional integrity. In the case of self-healing by\nreversible associative covalent bonds, Capelot et al. 41 developed a self-healing epoxy vitrimer capable of recovering\nnearly 77% of its original strength at 150 °C in the presence\nof transesterification catalysts, though embedding catalysts in the\nsystem might result in toxicity and also induce catalyst instability.\nKrishnakumar et al. 40 stated that healing\nepoxy for nearly 90% of the original strength was possible by disulfide\nbonds, which require a heating process of the crack region to activate\nthe bond exchange mechanism. The glass transition temperatures were\naround 60 °C, which prohibited the activation of disulfide exchange\nup to that temperature. Li et al. 42 also\ndeveloped healing epoxy materials, but they also needed heat to repair\nthem. Moreover, it is quite difficult to recover polymers by heating\ndue to their low thermal conductivity. To eliminate such limitations\nand extra steps, this paper proposes a new approach promising good\nlong-term structural stability and ease of repair. To the best of\nour knowledge, catalyst-free epoxy-based healing materials healable\nat room temperatures without any stimuli have not been reported yet. Meanwhile, research works have been focused on self-healing materials,\nespecially on vitrimers as representative polymers, which can heal\nintrinsically by covalently adaptable networks in the molecular structure.\nIn other words, vitrimers own a network with exchangeable bonds so\nthat the materials can be healed through the exchange reaction after\ndamage. Another prominent property of the vitrimer is the decrease\nin its viscosity as the temperature increases, obeying the Arrhenius\nlaw. A material can be called a vitrimer when the aforementioned properties\nare satisfied. Materials meeting only one of these requirements are\nclassified as vitrimer-like materials, 43 and this study developed vitrimer-like self-healing materials with\nthe disulfide bond in this respect. Among the vitrimer or vitrimer-like\nmaterials, many researchers have tried to develop self-healing vitrimers\nwith the disulfide bond, 44 − 60 which is because the disulfide bond reaction is facile to fabricate\nand promises high self-healing efficiency. Most of the self-healing\nmaterials with disulfide bonds, however, require external stimuli\nsuch as light, 44 − 49 heat, 50 − 55 catalyst, 56 − 58 pH, 59 and pressure. 60 As mentioned earlier, dynamic sulfur chemistries\nare highly adopted\nin self-healing polymers, including but not limited to thiolate/nanoparticle\nexchange, aromatic disulfide exchange, and gold(I)–thiolate/disulfide\nexchange reaction. 61 Among them, an aromatic\ndisulfide bond has been preferred widely to develop room-temperature\nhealable materials by a disulfide exchange reaction, which can take\nplace at room temperatures. 17 , 18 Apart from that, a\nhydrogen bond has also been used as a self-healing agent to introduce\nthe healing capability to the material. Many studies show that hydrogen\nbonds can initiate self-healing because of their reversible bond characteristics. 6 , 17 , 62 Specifically, Rekondo et al.\ndeveloped self-healing materials with disulfide and hydrogen bonds,\nbut it is worth noting that these materials are fabricated with TPU. 17 As mentioned earlier, TPUs are not ecofriendly\ndue to the utility of solvents, and they require a complicated synthesis\nprocess. Therefore, this paper differs from the literature as the\naim is to synthesize epoxy-based materials healable at room temperature\nwithout any external stimuli via disulfide and hydrogen bonds. These\nself-healing materials do not cause chemical waste generated from\nthe solvent and the catalyst, implying their facile process. Herein, to develop epoxy-based materials healable at room temperature\nwithout external stimuli, diglycidyl 1,2-cyclohexanedicarboxylate\n(DGCHD) was added to employ a hydrogen bond with a carbonyl group\nof ester. This hydrogen bond made the elastomer self-healed efficiently,\nas proven by Fourier transform infrared spectroscopy (FTIR) and a\ncontrol experiment. Poly(propylene glycol) diflycidyl (PPGDG), referred\nto as a soft segment, was added to epoxy resin to activate the disulfide\nexchange reaction by decreasing the glass transition temperature ( T g ). The reason why low T g is required is mainly that the disulfide exchange reaction\noccurs at temperatures lower than T g ,\nwherein decreasing the T g below room temperature\nresults in self-healing materials with elastomeric properties. The\nsoft segments were reported before as they promote a higher healing\nperformance, especially with the dense network, four-branched network. 63 Meanwhile, another study on thermoplastic polyurethane\n(TPU) showed that crystallinity and tightly packed segments affect\nself-healing behavior because the hard segment of TPU limits the segmental\nmotion and self-healing performance. 18 According\nto the TPU study, in this research, PPGDG was used to avoid crystallinity\ninstead of poly(ethyelene glycol) diglycidyl ether (PEGDG), and 2-aminophenyl\ndisulfide (2-AFD) was introduced due to a loosely packed segment instead\nof 4-aminophenyl disulfide (4-AFD). Control experiments were conducted\nto explore the effect of crystallinity and the tightly packed segment\nin epoxy. This paper conducted synthesis, characterization,\nself-healing\ntest, and control experiments of the S-vitrimer. First, the S-vitrimer\nself-healed at room temperature was synthesized and characterized\nwith FTIR, thermogravimetric analysis (TGA), and differential scanning\ncalorimetry (DSC). Then, a tensile test was conducted to measure the\nhealing efficiency at varying times and temperatures. The S-vitrimer\nshowed improved self-healing performance as the healing time and temperature\nincreased. Herein, it should be noted that self-healing occurs at\nroom temperatures without additional external heating since the glass\ntransition temperature ( T g ) of the produced\nepoxy vitrimer-like materials is lower than the room temperature.\nFinally, control experiments were carried out to investigate factors\ninfluencing self-healing behavior. It can be concluded that the disulfide\nand hydrogen bonds promoted self-healing, while the self-healing behavior\nwas hindered by the crystallinity of the soft segment and tightly\npacked segments in the epoxy vitrimer-like materials.",
"discussion": "3 Results and Discussion 3.1 Fabrication and Characterization of Epoxy\nVitrimer-Like Materials As shown in Figure 2 , elastomeric epoxy vitrimer-like materials\nnamed the S-vitrimer with a self-healing ability at room temperature\nwere developed. 2-AFD was used to introduce the aromatic disulfide,\nwhich made the S-vitrimer self-healable at room temperature. DGCHD\nwas added to employ the hydrogen bond formed by the carbonyl and hydroxyl\ngroup, which assists the aromatic disulfide in recovering the properties\nof the S-vitrimer. The intrinsic effect of disulfide and hydrogen\non self-healing is examined thoroughly in Section 3.3 . On the other hand, the main goal of PPGDG\nin the system was to decrease the T g of\nthe cured epoxy due to the linear aliphatic structure of PPGDG, promoting\nrelatively higher mobility. Briefly, lower T g was expected to increase mobility and hence the rate of self-healing. Figure 2 Synthesis\nof the self-healing epoxy disulfide vitrimer-like material\n(S-vitrimer). The vitrimer is healed through a disulfide exchange\nreaction and a hydrogen bond between carbonyl and hydroxyl groups. FTIR analysis was performed to observe the chemical\nstructure of\nthe synthesized S-vitrimers. The structure of the S-vitrimer can be\npredicted by observing the range of spectra corresponding to the epoxide\nring because the curing reaction of epoxy occurs through an epoxide\nring-opening reaction. To check the epoxide ring, 890 to 950 cm –1 spectra and 3300 to 3500 cm –1 were\ninvestigated. As shown in Figure 3 a, the reduction of 890 to 950 cm –1 peaks was confirmed in FTIR spectra. Also, the appearance of the\n3300 to 3500 cm –1 peak is shown in Figure 3 a. A reduction of the 890 to\n950 cm –1 area appeared due to the epoxide ring-opening\nreaction, and the appearance of a 3300–3500 cm –1 wide peak indicates the hydrogen bond of the hydroxyl group. This\nresult showed that the S-vitrimer was cured, considering the ring-opening\nreaction of the epoxide ring. Although the epoxide ring peak (890\nto 950 cm –1 ) did not disappear completely, it seems\nthat the reaction was finished. Comparing FTIR spectra of the S-vitrimer\ncured for 30 h with the S-vitrimer cured for 15 h, the spectra of\nthe 30 h-cured S-vitrimer are very similar to 15 h-cured, as shown\nin Figure 3 b, which\nmeans that the vitrimer-like material no longer reacted after 15 h\ncuring. DSC isothermal analysis also showed that the curing reaction\nwas completed at 150 °C. If there were still epoxides remaining\nto react in the S-vitrimer, an exothermal peak would be observed.\nHowever, no exothermic behavior was found when the S-vitrimer was\nleft at 150 °C for 1 h in DSC, as shown in Figure 3 c. The S-vitrimer did not show any thermal\nbehavior at 150 °C, which means that the curing reaction of the\nS-vitrimer did not occur anymore after 15 h curing. Figure 3 b,c proves that the curing\nreaction of the S-vitrimer was completed at 150 °C. Figure 3 Curing analysis\nof the S-vitrimer. The FTIR graph of (a) uncured\n(red trace) and 15 h-cured (black trace) S-vitrimers and (b) 15 h\n(black trace)-cured and 30 h-cured (orange trace) vitrimer-like materials.\n(c) Isothermal DSC analysis of the S-vitrimer at 150 °C. Both\n(b) and (c) results suggested that the curing reaction was completed. It is difficult to measure the degree of curing\n(α) in FTIR\nby simply comparing the absolute absorbance value because the shape\nof the spectrum changes after the ring-opening reaction. Therefore,\nthe peak should be normalized with respect to the reference peak that\nis not engaged in the curing reaction. In this paper, the ether bond\nwas used as a reference peak because the ether bond does not attend\nthe curing reaction. The degree of curing (α) was calculated\nby measuring the height of the peak as described in detail. First,\nfind the epoxide ring peak (890 to 950 cm –1 ) and\nthe ether bond peak (1000 to 1100 cm –1 ). Next, the\nbaseline was drawn between the local minimum, and the height of the\npeak was measured by gauging the height between the peak point and\nthe baseline ( Figure S1 ). Then, the degree\nof curing can be evaluated as in eq 2 2 where h before epoxy is the height of the\nepoxide ring before the curing reaction, h before ether is the\nheight of the ether bond before the curing reaction, h after epoxy is\nthe height of the epoxide ring after the curing reaction, and h after ether is the height of the ether bond after the curing reaction. As presented\nin Table S1 , the degree of curing for the\nS-vitrimer is found as 47.2%. The aforementioned curing behavior\nof the vitrimer-like material\ncan be attributed to the steric hindrance of PPGDG. To prove this\neffect, a 2-AFD hardener was cured with only one of the resins among\nDGEBA, PPGDG, and DGCHD, which were named E-vitrimer, P-vitrimer,\nand H-vitrimer, respectively. In the case of the E-vitrimer and H-vitrimer,\nit is observed that the peak between 890 and 950 cm –1 almost disappears, as shown in Figure S3 . By the same calculation method of the curing degree, it was found\nthat curing is completed 70 and 90% in the E-vitrimer and H-vitrimer,\nrespectively. In contrast, the 890 to 950 cm –1 peak\nof the P-vitrimer remained still noticeable after curing, and the\ndegree of curing ended at only 26% ( Table S1 ). It is concluded that the long-chained structure of PPGDG increases\nsteric hindrance and hampers the reaction. The decrease in the degree\nof reaction is attributed to the S N 2 reaction during epoxy\ncuring, 64 , 65 which is significantly affected by steric\nhindrance, as shown in Figure 4 . It is the reason why the epoxide ring stretching peak did\nnot disappear completely in the S-vitrimer, although the reaction\nended at 150 °C. Figure 4 Mechanism of the epoxide ring and amine curing reaction. 65 The reaction occurs through the S N 2 reaction, whose reactivity is mainly affected by steric hindrance.\nReprinted with permission from ref ( 65 ). Copyright 2007 ACS Publications. TGA experiments were carried out from 40 to 600\n°C under a\nnitrogen and air atmosphere to measure the thermal stability in the\ninert and normal states. Thermal stability can be obtained by analyzing\nthe derivative weight, as shown in Figure 5 c,e. As shown in Figure 5 c, thermal degradation began around 200 °C,\nwhich means that the S-vitrimer should avoid being left around 200\n°C under air since it begins to degrade afterward. A 5% weight\nreduction was found at 256 °C and a 10% weight reduction was\nshown at 275 °C, as shown in Figure 5 d. The S-vitrimer started degrading most\nrapidly at around 332 °C. The thermal behavior of the S-vitrimer\nunder nitrogen was very similar to the case under air, as shown in Figure 5 e,f. It is inferred\nthat the S-vitrimer was degraded by heat rather than the external\nair effect. As shown in Figure 5 a, the T g value of the S-vitrimer\nis 5 °C by DSC analysis. As shown in Figure 5 b, the T g value\nof the S-vitrimer is 20 °C by DMA analysis. Generally, there\nis a difference in the T g value in DSC\nand DMA. It is because T g is not a specific\npoint like melting and boiling temperature, but it is a temperature\nrange between the first-order and second-order phase transition. The T g value changes according to various parameters\nsuch as the heating rate and frequency, leading to the difference.\nThe DSC is a static method where the energy difference is measured\nto identify the glass transition temperature because when the transition\noccurs, the heat capacity of polymers changes. On the other hand,\nit is also possible to analyze glass transition temperature through\nDMA in terms of molecular mobility because of the free volume increase\nin the T g value. It is a dynamic method\nby which the viscoelastic modulus is measured. The storage modulus,\nrelated to elasticity, decreases, while the loss modulus, related\nto viscosity, increases when heating in the glass transition temperature\nrange. It is because the polymer changes into the rubbery phase due\nto the polymer chain uncoiling phenomenon when the temperature is\nhigher than the T g value. Figure 5 Thermal behavior of the\nS-vitrimer. (a) DSC graph of the S-vitrimer.\n(b) DMA graph of the S-vitrimer. The black line corresponds to the\nstorage modulus, while the red line is the tan δ curve.\nThe TGA graph of the S-vitrimer under (c, d) air and (e, f) nitrogen.\nThe S-vitrimer began to thermally degrade at about 200 °C and\ndegraded rapidly at about 340 °C. The S-vitrimer began to thermally\ndegrade at about 200 °C and degraded rapidly at about 340 °C.\nThe black line is the weight percent, and the red line is the derivative\nweight. 3.2 Self-Healing Test To confirm the\nperformance of self-healing, self-healing experiments were conducted\nas follows. The S-vitrimer was cut into two pieces, and then two pieces\nwere directly put into contact at room temperature without any external\nforce. The reattached S-vitrimer was left at room temperature for\nvarious durations, and strength was measured through the tensile test.\nHealed strength obtained by the tensile test was compared with pristine\nstrength to evaluate the self-healing efficiency of the S-vitrimer,\nas shown in Figure 6 . Figure 6 Healing behavior of the S-vitrimer. The stress–strain curve\nof the S-vitrimer (a) healed at room temperature for varying times.\nThe S-vitrimer is healed 26, 41, 58, and 79% after 24, 48, 72, and\n96 h, respectively. (b) Healed at varying temperatures for 24 h. The\nS-vitrimer is healed 28, 33, 42, and 46%, respectively. (c) Healed\nat 60 °C and showed an 80% healing efficiency after 48 h. (d)\nS-vitrimer was cut with a blade and then healed at room temperature.\nThe healed S-vitrimer was left at room temperature, and then (e) tensile\ntest with a 50 mm/min loading speed was performed. The S-vitrimer showed 26, 41, 58, and 79% healing\nefficiencies\nwhen healed for 24, 48, 72, and 96 h, respectively, as shown in Figure 6 a. These results\ndemonstrated that healing efficiency increases as the healing time\nincreases. The longer healing time means that the time for the disulfide\nexchange reaction increases. It confirms that as the S-vitrimer undergoes\na disulfide exchange reaction for a longer time, the S-vitrimer heals\nitself more. For instance, the healing efficiency of the S-vitrimer\nreached about 80% after healing for 96 h at room temperature. The S-vitrimer can be healed at room temperature due to the aromatic\ndisulfide bond and the hydrogen bond. Herein, the aromatic disulfide\nbond exists in 2-AFD, and the hydrogen bond comes from the hydroxyl\ngroup and carboxylic group of DGCHD. The hydroxyl group is formed\nas a result of the epoxide ring-opening reaction. The role of the\naromatic disulfide bond and the hydrogen bond in healing is investigated\nin detail in Section 3.3 . The aromatic disulfide bond undergoes an exchange\nreaction at room\ntemperature, even in a solid state. It leads to an exchange reaction\nin a low-energy environment like room temperature. There are two reasons\nwhy aromatic disulfide exchange reactions can occur at room temperature.\nFirst, the sulfenyl radical created when the exchange reaction occurs\nis stabilized due to the delocalization of the free radical 66 , 67 ( Figure 7 ). Additionally,\ndue to the increase in the number of antibonding electron quicks in\nmolecular orbitals, dissociation of the S–S bond occurs more\nquickly, making exchange reactions occur at room temperature. 66 The working mechanism of the aromatic disulfide\nexchange reaction in the healing process can be explained as follows.\nThe aromatic disulfide exchange reaction works as a chemical operator\nso that the chemical bonds broken under fracture can be reconnected\nand recovered to their original state. As a result of these recovered\nbonds at the fracture surface, the material state can go back to its\noriginal properties. 17 , 18 Figure 7 [2 + 1] radical-mediated reaction mechanism\nof the disulfide exchange\nreaction. 67 The radical is stabilized due\nto the delocalization of the radical electron caused by the aromatic\nring bonded to sulfide. (a) Aromatic disulfide structure before the\nexchange reaction. (b) Aromatic disulfide exchange reaction through\nhemolysis. (c) Result of the exchange reaction. Reprinted with permission\nfrom ref ( 67 ). Copyright\n2016 Elsevier. To initiate the disulfide exchange reaction, the\nglass transition\ntemperature ( T g ) of the aromatic disulfide\nvitrimer must be lower than room temperature because the disulfide\nexchange reaction can occur when the vitrimer has enough mobility. 68 Mobility allows the disulfide bond exchange,\nowing to the collision between S atoms, as there would be no collision\nin the molecule at a temperature lower than T g . Through DSC and DMA, it is confirmed that T g of the S-vitrimer is lower than room temperature, which\nmeans that the disulfide exchange reaction can take place at room\ntemperature as well as the healing process. As a result of DSC analysis,\nit is found that the T g value of the S-vitrimer\nis 5 °C, as shown in Figure 5 a. Also, through the DMA experiment, the T g value of the S-vitrimer is detected as 20 °C, as\nshown in Figure 5 b.\nWhen the S-vitrimer was left in the refrigerator at 5 °C, self-healing\nbehavior was not observed since the disulfide exchange reaction did\nnot get activated below T g . The\nhydrogen bond also allows the S-vitrimer to be healed at room\ntemperature efficiently within their weak bonding structure. It is\neasy to break and reform hydrogen bonds reversibly, allowing a more\nefficient and easier healing mechanism. As shown in Figure 3 a, a 3300–3500 cm –1 wide peak does not exist before curing. After curing,\na wide peak appears in the range of 3300–3500 cm –1 . The formation of a broad peak around 3300–3500 cm –1 infers that a hydrogen bond was created after the curing reaction. In addition, the S-vitrimer was healed at different temperatures\nto understand the effect of temperature on the healing efficiency\nbetter. The S-vitrimer showed 28, 33, 42, and 46% healing efficiencies\nat room temperature, 40, 50, and 60 °C, as shown in Figure 6 b. The increased\nhealing efficiencies are attributed to the increased material mobility.\nAs the temperature increases, the mobility also increases, leading\nto more efficient healing of the S-vitrimer. It should be pointed\nout that the healing efficiency reaches nearly 80% after healing for\n48 h at 60 °C, as shown in Figure 6 c. Furthermore, the S-vitrimer showed such a high healing\nefficiency at 60 °C in a shorter time than the same healing process\nat room temperature. 3.3 Control Experiments Three control\nexperiments were performed by changing one parameter each time to\ninvestigate the key factors that affect self-healing behavior. All\ncontrol groups were cured for 15 h at 150 °C, and the curing\nreaction was followed by FTIR analysis, as shown in Figure 8 f. After the curing reaction,\n890 to 950 cm –1 peaks remained in C1, C2, and C3,\nsimilar to the S-vitrimer, and the degree of curing was also found\nto be similar ( Table S1 ). Within the similar\ndegree of curing of the S-vitrimer and control samples, it is deduced\nthat the healing behavior of these control groups can be compared\nwith the S-vitrimer. The control groups were also analyzed under isothermal\nDSC analysis ( Figure S4a,c,e ) and did not\nshow any exothermic peak corresponding to the further curing reaction.\nSuch a result indicated that the curing reaction of three control\ngroups was completed at 150 °C. In other words, the control groups\nwere also not fully cured, similar to the S-vitrimer. Self-healing\nexperiments of three control groups were performed identically to\nthe process mentioned above, and all self-healing experiments were\nconducted at room temperature for 96 h. Figure 8 (a) Synthesis and starting\nmaterials of control group C1 without\ndisulfide. (b) Stress–strain curve of C1, which showed a 6.4%\nhealing efficiency. (c) Synthesis and starting materials of control\ngroup C2 without a pendant group of the soft segment. (d) Stress–strain\ncurve of C2, which showed a 45% healing efficiency. (e) Synthesis\nand starting materials of control group C3 with a para-substitution\nhardener. C3 did not show self-healing behavior. (f) FTIR graph of\ncontrol groups. The graph shows 890 to 950 cm –1 corresponding\nto the epoxide ring that remained in C1 (green trace), C2 (black trace),\nand C3 (red trace). First, epoxy materials without disulfide (C1) were\nsynthesized\nto explore the disulfide and hydrogen bond effect. C1 was fabricated\nusing an EDA hardener instead of a 2-AFD hardener used in the S-vitrimer\n( Figure 8 a). It is\npossible to study both disulfide and hydrogen bond effects with EDA\nbecause it has a CH 2 –CH 2 bond instead\nof a S–S bond in AFD. The result of the C1 experiment\nshowed a 6.4% healing efficiency\n( Figure 8 b). This result\nindicated that the absence of disulfide led to a major reduction in\nself-healing behavior. At the same time, it indicated that hydrogen\nbonds play a role in self-healing since there is still healing in\nthe C1 sample with only hydrogen bonds in its structure. Hence, the\nhydrogen bond is found to be capable of performing self-healing in\nthe absence of disulfide bonds, wherein the efficiency is much less\nthan the S-vitrimer due to the absence of disulfide bonds. The hydrogen\nbond effect was hereby examined through both FTIR results ( Figure 3 a and control group\nC1). Meanwhile, DSC analysis showed that C1 has a T g value of 15 °C lower than room temperature ( Figure S4b ). Next, PEGDG was employed instead\nof PPGDG to investigate the effect\nof the pendant group of the soft segment, which is related to crystallinity\n( Figure 8 c). As shown\nin Figure 8 c, PEGDG\ndoes not have a CH 3 substituent that exists in PPGDG. It\nwas possible to explore the effect of methyl substituent branching\nby comparing PPGDG and PEGDG, as they both have about eight repeating\nunits. Moreover, the T g value of C2 is\naround 6 °C ( Figure S4d ), which is\ncomparable to the T g value of the S-vitrimer.\nTherefore, the only difference between the S-vitrimer and C2 is the\nsoft segment unit, PPGDG, and PEGDG. It was found that the healing\nefficiency of C2 was only 45% ( Figure 8 d), implying the\nimportance of the extra methyl substituent of PPGDG on self-healing\nperformance. Such a decrease in the healing efficiency of C2 is attributed\nto the pendant group 18 of the resulting\nepoxy vitrimer-like materials, as PEGDG has higher crystallinity than\nPPGDG due to the substituent. It was reported that there is a chain\nbranching effect in polyethylene, 69 such\nthat the more methyl substituent polyethylene has, the lower crystallinity\nis observed in the structure, as shown in Scheme S8 . To verify the existence of a pendant group, the 1700\nto 1750 cm –1 peak corresponding to the carbonyl\ngroup was analyzed\nthrough attenuated total reflection-Fourier transform infrared spectroscopy\n(ATR-FTIR) ( Figure 9 a). A peak at 1732 cm –1 is observed, corresponding\nto free carbonyl without a hydrogen bond with a hydroxyl group. The\npeak at 1718 cm –1 is related to the hydrogen-bonded\ncarbonyl group. 70 The carbonyl region of\nthe FTIR spectrum is resolved into free and hydrogen-bonded carbonyl\nto compare the number of the carbonyl group with and without hydrogen\nbonds by evaluating the area below the curves ( Figure 9 b,c). The results exhibited that the number\nof the carbonyl group with hydrogen bonds reached 87% of free carbonyl\nin the S-vitrimer. In the case of C2, the number of the hydrogen-bonded\ncarbonyl group just ended up at 66.0% of the free carbonyl group ( Table S3 ). This is attributed to the soft segment\nof the S-vitrimer having a CH 3 pendant group, resulting\nin less crystallinity, which promotes the hydrogen bond. As a result,\nthe S-vitrimer having higher healing efficiency than C2 signifies\nthe role of hydrogen bonds. Figure 9 Molecular structure of control vitrimer-like\nmaterials. (a) ATR-FTIR\nspectra of the S-vitrimer and the C2 experiment. In the spectra, 1732\ncm -1 corresponds to free carbonyl (without interaction\nwith the hydroxyl group) and 1718 cm -1 corresponds\nto hydrogen-bonded carbonyl. The carbonyl region of the FTIR spectrum\nis resolved into free and hydrogen-bonded carbonyl peaks in (b) C2\nand (c) S-vitrimer. The molecular structure of (d) S-vitrimer, which\npossesses a bent structure of a hardener, has higher free volume and\nloosely packed segment. (e) C3 (right), which possesses a quasi-linear\nstructure of a hardener, has lower free volume and a tightly packed\nsegment. To ensure the effect of the hardener, another control\nexperiment\nwas conducted. In this last control experiment, 2-AFD ortho -substitution was replaced with 4-AFD para -substitution\n( Figure 9 e). The DSC\nresult showed that C3 has a T g value of\n25 °C ( Figure S4f ). Self-healing experiments\nwere conducted at 60 °C as well as at room temperature. Even\nthough 60 °C is far higher than T g in which C3 has enough mobility, there was no self-healing behavior\nobserved similar to the case at room temperature. It can be explained\nthat tightly packed segments hindered self-healing behavior regardless\nof mobility. Kim et al. also reported that tightly packed segments\naffect self-healing behavior in a negative way. 18 As shown in Figure 9 d,e, an ortho -substituted hardener with a\nbent structure generates a loosely packed conformation, while a para -substituted hardener with a quasi-linear structure\nlets molecules be tightly packed. Therefore, it clarifies the lower T g value of the S-vitrimer ( Table S2 ) as it is less packed and has more free volume compared\nto C3. In summary, it is concluded that a highly packed structure\nprevents the healing mechanism in self-healing materials."
} | 8,738 |
21731670 | PMC3123342 | pmc | 6,003 | {
"abstract": "Cleaning behaviour is deemed a mutualism, however the benefit of cleaning interactions to client individuals is unknown. Furthermore, mechanisms that may shift fish community structure in the presence of cleaning organisms are unclear. Here we show that on patch reefs (61–285 m 2 ) which had all cleaner wrasse Labroides dimidiatus (Labridae) experimentally removed (1–5 adults reef −1 ) and which were then maintained cleaner-fish free over 8.5 years, individuals of two site-attached (resident) client damselfishes (Pomacentridae) were smaller compared to those on control reefs. Furthermore, resident fishes were 37% less abundant and 23% less species rich per reef, compared to control reefs. Such changes in site-attached fish may reflect lower fish growth rates and/or survivorship. Additionally, juveniles of visitors (fish likely to move between reefs) were 65% less abundant on removal reefs suggesting cleaners may also affect recruitment. This may, in part, explain the 23% lower abundance and 33% lower species richness of visitor fishes, and 66% lower abundance of visitor herbivores (Acanthuridae) on removal reefs that we also observed. This is the first study to demonstrate a benefit of cleaning behaviour to client individuals, in the form of increased size, and to elucidate potential mechanisms leading to community-wide effects on the fish population. Many of the fish groups affected may also indirectly affect other reef organisms, thus further impacting the reef community. The large-scale effect of the presence of the relatively small and uncommon fish, Labroides dimidiadus, on other fishes is unparalleled on coral reefs.",
"introduction": "Introduction On coral reefs, cleaning organisms - which include shrimps and fishes - perform the function of removing ectoparasites from ‘client’ organisms, usually reef fishes [1] . Cleaning behaviour has been used as a classic example of mutualism and, recently, to test cooperation theory [2] . Surprisingly, the health benefit to clients, in terms of body size, has never been measured [3] nor have any mechanisms involved in effects on fish communities [4] , [5] been elucidated. On Atlantic and Indo-Pacific coral reefs, cleaner fishes interact with many client fish species [5] – [7] . The most common Indo-Pacific cleaner fish, Labroides dimidiatus \n [8] , inspects an average 2297 clients day −1 and consumes an average 1218 ectoparasites day −1 \n [9] . Individual clients are often cleaned repeatedly, some up to 144 times day −1 \n [10] . Cleaner fishes often reside in ‘cleaning stations’ [3] ; this site fidelity makes them an ideal model system for the study of localised effects of cleaning interactions. There has been considerable debate about the mutualistic nature of cleaning symbioses. Benefits to cleaners are well documented; cleaners enjoy nutritional rewards from eating ectoparasites and protection from predation [3] . The benefit of cleaning to clients, however, remains contentious. Fish parasites can lower host growth, recruitment, and fecundity, and increase mortality [11] , [12] . They have also been shown to affect fish foraging, swimming, and anti-predator behavior [13] . Thus, variation in parasite loads can lead to changes in their host community. However, early experimental removals of cleaner fish found no effects on ectoparasite or fish numbers after the removal of L. phthirophagus for one and seven months and L. dimidiatus for six months and two years [14] – [18] . In contrast, the removal of L. dimidiatus affected clients in three experiments. A short-term study (24 h and 12 d) at Lizard Island found that caged Hemigymnus melapterus had more and different sizes of parasitic isopods in the absence of cleaners, compared with controls [19] , [20] . After 4–20 months, in the Red Sea, the species richness of ‘visitor’ (fish species that can move between patch reefs) and ‘resident’ (site-attached fish species) clients were reduced; however, fish abundance was not measured [4] . After 18 months, at Lizard Island, the species richness and abundance of visitors were reduced; however, no effect on resident species richness was detected [5] . A reduction in visitors could simply involve a change in visitation rates to reefs; in residents, the presumption is that it is more likely due to lower survivorship or recruitment [5] . Whether cleaner fish affect resident abundance over the long term (>6 months) or affect juveniles, however, has never been examined. Most importantly, the effect of cleaning on client fish fitness, including fish size, a common measure of condition and growth in fishes [21] , has never been measured. We investigated the long-term effects of cleaners on fish communities at Lizard Island in the longest study of its kind. We used an ongoing study in which patch reefs at two sites had been kept free of L. dimidiatus for 8.5 years, while similar control reefs had not had cleaner fish removed. First, to determine whether cleaning affects client size, we surveyed two common resident client fishes, Pomacentrus moluccensis and P. amboinensis, whose life spans are around eight (Shalan-Louise Bray, unpub. data) and six years (Mark I. McCormick, unpub. data.), respectively. Therefore, many individuals had experienced these experimental conditions for their entire lives. For each of these two species on each reef, we measured the sizes of all individuals and their total abundance. Second, we recorded the abundance and species richness of residents, juvenile visitors, and adult visitors on experimental removal and on control reefs.",
"discussion": "Discussion Over an 8.5 year period, the removal from patch reefs of a single species - the cleaner wrasse Labroides dimidiatus - shifted the size distributions of two resident damselfish populations toward smaller individuals. This is the first demonstration that individual clients gain a fitness advantage from cleaners in the form of increased size. Cleaner absence also reduced the abundance and species richness of resident species and adult visitor species and the abundance of juvenile visitors. We argue these findings suggest that cleaner fish presence affects, directly or indirectly, the growth, survivorship, and/or recruitment of coral reef fishes; this is a first demonstration of potential mechanisms by which cleaners affect fish communities. To date, studies on the removal of key functional groups from coral reef fish communities have largely focused on the effects of large, mobile herbivores or predators due to their rapid worldwide depletion through human exploitation [22] – [24] . Here we demonstrate the dramatic impact of removing a single fish species that is small (maximum 8 cm total length) and not very abundant (1–5 adults per reef; mean±s.e. reef area: 131±34 m 2 , Table S5 ), but is nonetheless of great ecological importance. Cleaner fish remove ectoparasites from client fishes [1] ; therefore, the profound influence that this species had on the local fish community indicates the powerful influence that ectoparasites have on coral reef fishes. After 8.5 years, the size frequency distributions of the damselfishes Pomacentrus moluccensis, and of P. amboinensis when two reefs with very few individuals were omitted, were shifted towards smaller individuals on reefs without L. dimidiatus . In contrast, after the preliminary removal of L. dimidiatus there had been no difference in the estimated mean size of P. moluccensis per reef between treatments after 3 and 6 months. One likely consequence of this decreased size after 8.5 years is a decreased number and size of reproductively active adults per reef. Since female size and fecundity are highly correlated in damselfishes [25] , reproductive output should be decreased on reefs without cleaner fish. P. moluccensis and other damselfishes are cleaned relatively infrequently compared with other clients included in the study [10] , suggesting that any benefits of cleaning may be more pronounced in other, more frequently cleaned or heavily parasitised species. We did not find an effect of cleaner fish presence on the abundance of P. moluccensis or P. amboinensis. This suggests that the smaller size of individuals in the absence of cleaners is not due to factors that increase abundance, such as increased post-settlement migration, a behaviour that is also rare in these damselfishes [26] , nor to increased recruitment. It is possible that the populations of these species are constrained more by habitat and social dynamics as they live in corals [27] and in small social groups [28] , respectively. Complex interactions between larval recruits and adults [29] and the large variation in recruitment events [26] may have also obscured any effect of cleaner fish presence. Multiple opposing indirect effects may also be acting concurrently; for example, the reduction in visitors (which included piscivores) on reefs without cleaner fish may increase prey survival. Since the abundances of P. moluccensis or P. amboinensis were not affected by cleaner presence, the shifts in size distributions may have been due to decreased rates of growth where cleaner fish were absent. Indeed, in the absence of cleaners, P. moluccensis individuals had a lower growth rate and more parasitic copepod juveniles but this occurred only in larger individuals [30] . The risk of infection with other clients' ectoparasites may also be higher on reefs without cleaners if other clients are also more parasitized on such reefs [14] , [19] , [20] . The changes in size distributions of P. moluccensis and P. amboinensis are potentially also the consequence of indirect effects on fish health. For example, aggression from a piscivore towards nearby fish is reduced in the presence of L. dimidiatus \n [31] ; this could, in turn, increase prey growth. This is the first time the presence of a cleaner organism has been shown to benefit (in terms of size) client individuals and confirms that cleaning is indeed a mutualism at this location, providing a firm foundation for studies of cooperation using this system (e.g. [2] ). While the species richness of resident fish (mostly damselfishes) per reef was not affected by cleaner presence after 18 months, it was lower after 8.5 years on reefs without cleaners. These results suggest that this effect of cleaners became apparent during this period. After 8.5 years, residents were also 37% less abundant, a parameter never previously measured. That the abundances of P. moluccensis and P. amboinensis were not affected by cleaner presence, however, indicates that only the abundance of some species was affected. Most resident species cannot and will not move readily to another part of the reef or patch reef to seek cleaning if no cleaning stations are available in their home range [32] . For these species, the benefits of being cleaned are perhaps not greater than the costs of traveling to a cleaner, which may include increased predation risk and energy output and loss of territory. On our isolated experimental reefs, swimming to another reef would involve a very high predation risk. Furthermore, the costs of not being cleaned may be lower for residents because they are smaller, with fewer and different ectoparasites compared to larger fishes [33] . Thus, the impact of cleaner fish removal may be less immediate in such fish and may only become detectable over a longer period. Consequently, changes to the community structure of resident fishes are likely not due to migration, but other factors, including reduced recruitment and mortality, associated with increased parasitism in the absence of cleaners but also unknown indirect effects. However, Bshary [4] found a reduction in the species richness of residents when L. dimidiatus was removed from reefs in the Red Sea, which was detected after 4 to 20 months. In Bshary's study, reefs were smaller (volume: 0.8 to 22 m 3 ), the species composition of clients was different, and species richness was lower, factors which all may contribute to how quickly an effect of cleaning is observed. Furthermore, abundance was not measured in the Red Sea, so it is possible that individuals of absent species were replaced by individuals from the remaining species. Finally, Simpson's diversity index did not differ with cleaner presence for residents in our study; this suggests that the relative abundances across resident species were relatively even, regardless of cleaner presence. This is the first study to consider juveniles separately from adult clients. The abundance of visitor juveniles was 65% lower in the absence of cleaners, suggesting that cleaner absence may decrease recruitment success or increase post-settlement migration of visitor species. Indeed, attacks by single parasitic gnathiid isopods decrease the successful settlement of P. amboinensis larvae (13mm, standard length), by affecting their performance as measured by swimming and oxygen consumption [12] , and many visitor juveniles settle at a similar size (A.S.G. pers. obs.). If such gnathiids are not removed from fish by cleaner fish or gnathiid population densities are higher on reefs without cleaner fish, this could result in a reduction in juvenile abundance. Dascyllus damselfish larvae can recognise the cleaner fish L. phthirophagus \n [34] . Therefore, if larvae select reefs because of the presence of cleaner fish, cleaner absence may reduce their abundance. Over the long term, these effects on juveniles could lead to a reduction in the number recruited to the adult population. Differential survival and habitat choice during settlement are well known in damselfishes (e.g. [28] ); however, the effect of cleaning remains unexplored. For adults of visitor species, local abundance and species richness were lower on reefs without L. dimidiatus compared with control reefs both after 18 months and 8.5 years. This indicates the pattern likely persisted during this period. After 8.5 yrs, the Simpson's diversity index was also affected by cleaner presence but this was related to reef area, with a decrease with decreasing reef area on reefs without cleaners and no association with area on controls. Since visitor species richness increased with area, regardless of cleaner presence, while abundance did not, the lower species richness on smaller reefs may have made the diversity of such reefs more vulnerable to cleaner absence. This pattern may be related to habitat diversity, which often positively affects species richness [35] . The observed shifts in both abundance and richness may be non-independent results if richness increases with abundance simply based on random expectations of sampling. Visitor clients, by definition, are more likely to modify their movements to search for cleaners, as these clients have the ability to select from several cleaning stations within their larger home ranges [4] , [5] . For adults, particularly of larger species, the impact of cleaner fish removal may be more immediate as larger fish have higher ectoparasite loads [33] . Indeed, parasitic isopods on a caged visitor ( Hemigymnus melapterus ) at this location were higher in the absence of cleaners after 24 h and 12 d [19] , [20] . Parasites are known to kill fish directly but might also do so indirectly by affecting metabolism [11] , [36] – [38] or behaviour [13] , [39] . Thus the decrease in visitor numbers could be due to parasite effects on survivorship. For both residents and adult visitors, when common species (see results for lists) were included as a random effect, there was still an effect of cleaners on abundance indicating the shift in species richness was not due to the loss of the more abundant fish species. The effects of cleaner fish on clients are unlikely to have been a temporary effect due to disturbance from the removal of L. dimidiatus shortly before the observations were made as this occurred only on two reefs, and involved only two juvenile cleaner individuals. More importantly, collecting cleaners was done quickly and controls were similarly disturbed by counting L. dimidiatus on most surveys and leaving collecting equipment on the reef during counts. The localised ecological effects of cleaner fish on fishes may have other indirect cascading effects on the reef community. Resident fishes consisted of herbivores and planktivores and visitors included herbivores, detritivores, piscivores, corallivores, and invertebrate predators. Herbivores were diverse and prominent on the reef (acanthurids, siganids, scarids, and some pomacentrids). The abundance of the most abundant and ubiquitous trophic group and family, the herbivorous Acanthuridae surgeonfishes ( Acanthurus, Ctenochaetus, Zebrasoma ), revealed abundance was 66% lower in the absence of cleaners. Herbivorous reef fishes limit the establishment and growth of algae that impede coral recruitment [40] and their removal has precipitated drastic shifts from coral to algal dominated systems [41] , [42] . Visitor species also included piscivores (lethrinids, lutjanids, haemulids, holocentrids, serranids), invertebrate predators (e.g. labrids), and corallivores (e.g. chaetodonts) [8] , [43] . Declines in the abundance of piscivores and invertebrate predators have been correlated with increases in fish prey abundance at fished sites [44] and have led to outbreaks of coral-eating starfish precipitating substantial declines in coral cover [45] , respectively. Corallivores slow the progression of black-band disease [46] . Indirect effects on the benthic composition of the reefs are also likely to have further effects on the coral reef fish community, including the diverse benthos-associated fish community [8] . A more detailed study of fish foraging behavior and benthic composition is clearly required on these reefs. The implications of this study are that (a) the behavioural interaction of cleaning of client fish by a relatively small number of small-sized fish has profound ecological consequences and (b) as ectoparasites are central to cleaning interactions, parasites can have a large effect on the population and community ecology of reef-fish. The presence of L. dimidiatus had remarkable effects on the local coral reef fish community that were considerably disproportionate to this species' small size and relatively low abundance. Potential mechanisms proposed for the above changes are effects on fish behaviour, movement, habitat choice, mortality, growth, and recruitment. Although this study measured only local effects, some effects may extend further. For example, the effect on the sizes of female fish, and hence the number of propagules produced [25] , might increase dispersal to other areas. Furthermore, the effects of cleaner fish were consistent at two sites, suggesting that the strong effect of cleaner fish presence may also apply to abundance estimates of fish at a larger scale. The dramatic declines in fish and fish species numbers caused by the removal of this single cleaner fish species are comparable with significant fishing pressure [45] , one of the leading known factors in coral reef community decline. Positive interactions, including mutualisms, are considered important to communities because they make the environment, directly or indirectly, more favourable for associated species which in turn often facilitates the establishment of other species [3] . At larger regional scales, positive interactions enhance diversity via an increase in habitat diversity [47] . In our case, client fish provide cleaner fish with nutrients, as plant-mycorrhizal fungi, zooxanthellae-coral, and plant-pollinator associations do for their fungi, coral, and pollinator partners [47] . In exchange, in each case the other partner enjoys a more favourable environment. This may directly increase fish biodiversity, but there are likely also other indirect cascading benefits, for example habitat modification by various fishes [8] , which may allow more species to coexist. \n L. dimidiatus is one of the top ten most exported aquarium fish species to the United States of America and the United Kingdom [48] . The ecological effects of the large scale removal of L. dimidiatus, however, are unknown. Given the importance of the species L. dimidiatus , conservation and management strategies may need to also focus on the protection of this key species."
} | 5,124 |
30002666 | PMC6032008 | pmc | 6,010 | {
"abstract": "Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro . Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant–endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H 2 DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant–endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype.",
"introduction": "Introduction Plant micropropagation in vitro has various applications in germplasm storage, industrial scale production of vegetatively propagated plants, plant biology research, and genetic transformations. However, the application of this method remains limited for recalcitrant plant species or genotypes, and several major agronomic crops still present a challenge ( Birch, 1997 ; Benson, 2000 ; Pence, 2010 ). One of the problems with in vitro cultivation is that plants are exposed to non-natural conditions, such as synthetic cultivation media, low irradiance, low CO 2 concentration during light periods, or high air humidity. These factors can lead to an imbalance in the plants physiological equilibria and stress ( Benson and Roubelakis-Angelakis, 1994 ; Cassells and Curry, 2001 ). The composition of plant growth regulators and/or mineral nutrients into the cultivation medium has been a main focus of studies designed to address the optimization of plant propagation methods in vitro ( Gaspar et al., 1996 ; Ramage and Williams, 2002 ). However, the possible utility of biological interactions with microorganisms to improve plant growth and stress tolerance in vitro has rarely been addressed. In nature, plants live in intimate association with microorganisms that help regulate the plant response to pathogens and environmental stresses ( Singh et al., 2011 ). Endophytic bacteria are a group of endosymbiotic microorganisms that live in plant tissues ( Schulz and Boyle, 2006 ), and the plant growth-promoting properties of endophytic bacteria have been extensively studied (see recent reviews by ( Xia et al., 2015 ; Miliute et al., 2015 ; Santoyo et al., 2016 ). In contrast, endophytic bacteria have been often regarded as contaminants of in vitro cultures ( Kulkarni et al., 2007 ; Ray et al., 2017 ). However, several studies have shown that bacterial endophytes are common in plant tissues grown in vitro and that their beneficial effects on plant growth indicate that they may be useful as growth-promoting agents. In previous studies, a succession of bacterial communities that colonized pineapple microplant organs in vitro were characterized ( Abreu-Tarazi et al., 2010 ) Similarly, endophytic bacteria were isolated from strawberry tissues cultivated in vitro ( Kukkurainen et al., 2005 ; Dias et al., 2009 ), and their beneficial effect on the acclimatization of the seedlings under greenhouse conditions was demonstrated ( Dias et al., 2009 ). Recently, the effects of bacterial endophytes in different in vitro culture phases and in different plant organs of Prunus avium were studied; isolates of the endophytes Rhodopseudomonas palustris and Microbacterium testaceum promoted rooting in two difficult-to-propagate genotypes ( Quambusch et al., 2016 ). Botta et al. (2013) demonstrated that Azospirillum brasilense and Gluconacetobacter diazotrophicus inoculated singularly or together conferred plant growth-promoting activity on tomato plants grown in vitro . Further, the drought stress reducing activity of endophytic Bacillus and Pseudomonas spp. strains was demonstrated in grapevine plants grown in vitro ( Salomon et al., 2014 ). The typical forms of plant–microbe interactions can be categorized into commensal, mutualistic, or pathogenic. However, many microorganisms exhibit different forms of relationships with plants during their life cycles ( Newton et al., 2010 ). It is proposed that at an initial stage, all microorganisms trigger an immune response in plants, while later events lead to the refinement of the interaction based on the capability of the microorganism to escape the host defense response ( Zamioudis and Pieterse, 2011 ; Hardoim et al., 2015 ). The early events involved in the formation of the plant–microorganism interaction stimulate complex signaling events that include characteristic intracellular accumulation of active oxygen and nitrogen compounds (ROS/RNS), which have also been documented for interactions involving endophytic bacteria ( Garcia-Brugger et al., 2006 ; Bordiec et al., 2011 ). Although eventually bacterial endophytes settle down as mutualistic colonizers, they may also prime the plant defense reactions and stress tolerance by inducing systemic resistance ( Zamioudis and Pieterse, 2011 ; Pieterse et al., 2014 ). Previously, bacterial strains of the genera Bacillus and Pseudomonas have been considered the most common groups to induce systemic resistance ( Kloepper and Ryu, 2006 ). Endophytic microorganisms provide potential means to reduce plant stress under in vitro conditions, which would allow for the extension of plant micropropagation techniques into recalcitrant plant genotypes. An outlying objective would be to understand the processes that lead to mutualistic endophyte colonization and the priming of the plant defense and tolerance to stress. In a previous study, we isolated 38 endophytic bacteria from apple buds of cultivars “Gala,” “Golden Delicious,” and “Orlovim” grown under field conditions ( Miliute et al., 2016 ). Biochemical analysis revealed several traits that are important for plant growth stimulation, including nitrogen fixation and the production of indole-3-acetic acid and siderophores that could have important implications on plant growth under in vitro conditions. Therefore, in this study, we assessed the effect of seven endophytic bacteria strains of Bacillus and Pseudomonas spp. on apple shoot biomass and auxiliary shoot propagation in vitro . Further, we used a model system consisting of an apple cell suspension to establish strain-specific effects during the initial stage of plant and microbe interaction, such as microbial cell adherence to plant cells and changes in the cellular redox balance. A proteomic analysis was employed to reveal differences in the expression of proteins participating in the apple cell response to bacterization with Bacillus spp. strains, each of which had a unique effect on the cellular redox balance and shoot development in vitro .",
"discussion": "Discussion Our study revealed contrasting effects of bacterization with selected endophytic bacteria strains of Bacillus and Pseudomonas spp. on apple shoot biomass accumulation and proliferation of auxiliary shoots in vitro . The stimulating or suppressing effect was not related to bacterial species or genus but varied depending on the specific bacterial strain ( Figure 1 ). The stimulating effect on shoot development indicates a mutualistic interaction between the bacteria and plant host; however, the cause of shoot growth suppression is not obvious. Since genotype-specific plant–endophyte interactions have been described for a number of plants ( Muller et al., 2015 ), it could be presumed that the cv. Gala shoots used in the study represented a fully compatible plant host for the strains of Pseudomonas spp. which were isolated from buds of the same cultivar grown in the field ( Miliute et al., 2016 ). However, two of the three strains of Pseudomonas spp. had negative effects on shoot development. Therefore, it appears that the reduced shoot growth and proliferation could be a consequence of altered interaction between plant host and endophytic bacteria under in vitro conditions. This agrees with previous observations that the outcome of the plant–endophyte interaction is determined not only by genetic background and physiological states of the plant host and microorganism but also on environmental conditions (see discussion by Partida-Martinez and Heil, 2011 ). As illustrated by the representative samples of auxiliary shoots shown in Figure 1 , suppressed shoot growth or proliferation was not associated with any adverse effect on shoot morphology, such as distorted morphology or tissue necrosis, that could be symptoms of microbial pathogenesis. In addition, no adverse effect on cell viability was observed during co-cultivation of the endophytic bacteria with the apple cell suspension. A previous study by Bordiec et al. (2011) showed that a challenge of grapevine cell suspension with a pathogenic Pseudomonas syringae pv. pisi strain induced a hypersensitive-like cell death response. In our study, the apple cell suspension maintained consistent morphology and viability level after a 6-h co-cultivation with endophytic bacteria. The mutualistic type of interaction between bacteria and apple shoots in vitro is further supported by the finding that all bacteria, including the three shoot growth suppressing strains Bacillus spp. Oa_4 and P. fluorescens Ga_3 and G_12, are able to reduce oxidative stress in the apple shoots ( Figure 2 ). This suggests that modulation of oxidative stress by endophytic bacteria might not be the only mechanism responsible for the observed differences in shoot growth. It is likely that the effect is related to bacterial capability to produce substances (e.g., phytohormones) that effect the vigor of plant growth or that the bacteria compete for resources required for plant growth. Bacillus and Pseudomonas genera include a number of common endophytic bacteria species, and plant growth-promoting traits have been characterized for different strains (see recent reviews by Miliute et al., 2015 and Santoyo et al., 2016 ). Our previous study established that several of the seven strains showed positive test results for atmospheric nitrogen fixation, nitrate reduction, and production of siderophores or indolyl acetic acid ( Miliute et al., 2016 ). Further, it was established that all seven strains involved in the study were able to maintain growth on minimal medium supplemented with ACC as a consequence of ACC deaminase activity (data not shown). Although direct parallels between these plant growth-promoting traits and the shoot growth-regulating properties established in the present study could not be clearly defined, there is a definite possibility that some of the traits could contribute to the shoot growth regulating effect. Plant cell suspensions have been used previously to study plant interactions with endophytes ( Bordiec et al., 2011 ; Boonsnongcheep et al., 2016 ) or rhizobacteria ( Verhagen et al., 2010 ). We used a similar experimental setup to characterize the interaction between the apple cells and endophytic bacterial strains. In the experiments with apple shoots, endophytic bacteria were inoculated at the base of the leaf petiole to perform bacterization of the shoots. Based on the studies carried out in planta , it could be presumed that the bacteria would migrate through the shoot tissues over an extended period of time, possibly days ( Compant et al., 2005 ). Such a gradual shoot colonization process would lead to an intricate sequence of perception, signaling, and response formation events in different parts of the shoot. To avoid such complexity, an apple cell suspension was employed as a model system. Although plant cells often form small clumps in the suspension culture, overall it presents a relatively homogeneous population of cells, and co-cultivation of apple cells with endophytic bacteria ensures a uniform response in the cell population. The model revealed distinct morphological, cellular redox balance, and protein expression differences characteristic to the apple and bacterial cell interaction. Bordiec et al. (2011) demonstrated that the endophytic bacteria Burkholderia phytofirmans strain PsJN associates with grapevine cells grown in suspension. Similar results were observed in our study. All endophytic bacteria strains showed a more pronounced association with the apple cells than did the E. coli control ( Figure 3 ), suggesting that association with plant cells could represent a common characteristic of endophytic bacteria similar to other plant growth-promoting bacteria in planta ( Rodriguez-Navarro et al., 2007 ). In contrast, the effect of the 6-h co-cultivation with endophytic bacteria on the intracellular redox balance of the apple cells was strain specific ( Figure 4 ). Interestingly, this effect at the early stage of the interaction was correlated with each strain’s capability to regulate shoot growth and proliferation during the 3-week co-cultivation. It was typical that apple cells incubated with shoot growth suppressing strains had reduced levels of DCF fluorescence compared to untreated cells, while fluorescence intensity increased or remained similar to the control in the cells co-cultivated with the shoot growth-promoting strains. Although several distinct processes have been shown to be involved in the oxidation of the H 2 DCFDA probe, several of the one-electron-oxidizing ROS/RNS contribute an important part ( Kalyanaraman et al., 2012 ). Considering this, our results agree with recent findings that showed a capability of endophytic bacteria to regulate ROS production. ROS were produced at early stages of rice root colonization by G. diazotrophicus PAL5 in planta ( Alqueres et al., 2013 ). Accumulation of ROS/RNS in plant cells bacterized with endophytic strains could be associated with early signaling processes involved in the plant response to microorganisms ( Garcia-Brugger et al., 2006 ) that further leads to activation of systemic resistance ( Pieterse et al., 2014 ). To further examine biological processes that lead to changes in the cellular redox balance at the early stage of plant–endophyte interaction and that may involve constituents responsible for shoot growth regulation, we assessed changes in the apple cell protein expression induced by the two Bacillus spp. strains Da_4 and Oa_4 that had contrasting effects on the cellular redox balance in the apple cells. Four distinct protein expression groups were identified by the cluster analysis of the protein expression data ( Figure 5 ) and were associated with similar biological processes involved in metabolism, protein metabolism, defense response, and cell development (Supplementary Figure 3 ). This suggests that both endophytic bacteria strains elicited responses related to defense and reorganization of cell development. However, very distinct expression patterns of the genes involved in these processes were revealed. The most notable such difference was the upregulation of HSC70-1 and PLAT1 after co-cultivation with Bacillus sp. strain Oa_4. Although the overall function of PLAT1 is poorly understood in plants, one role of the Arabidopsis PLAT1 gene has been revealed recently ( Hyun et al., 2014 ; Hyun et al., 2015 ). Overexpression of AtPLAT1 in Arabidopsis and tobacco has confirmed that the gene confers increased abiotic stress tolerance, including cold, drought, and salt stress, and that it promotes plant growth. Analyses of the AtPLAT1 promoter structure and in silico expression data suggest that the gene expression is regulated by ABA and that PLAT1 is a downstream target of the ABA signaling pathway ( Hyun et al., 2014 ). ABA is an important phytohormone that is involved in the regulation of plant water balance and plays a critical role in osmotic and other abiotic stress signaling ( Tuteja, 2007 ). It is known that the ABA response is mediated by ROS accumulation in plant cells ( Song et al., 2014 ). The role of ABA in endophytic bacteria-mediated stress tolerance in plants has been addressed by several studies ( Cohen et al., 2009 ; Salomon et al., 2014 ; Shahzad et al., 2017 ). One of the studies performed using grapevine plants grown in vitro reported that the drought stress-reducing activity of endophytic strains Bacillus licheniformis Rt4M10 and Pseudomonas fluorescens Rt6M10 was associated with the accumulation of high ABA levels in the leaves of the bacterized plants ( Salomon et al., 2014 ). In contrast, our results show that upregulation of PLAT1 by Bacillus sp. Oa_4 is most likely associated with the suppression of ROS/RNS production in apple cells and that it may also be related to reduced growth and proliferation of apple shoots in vitro . This suggests that under in vitro conditions, the function PLAT1 does not involve ABA- and ROS-mediated signaling. HSC70-1, the other protein significantly upregulated after co-cultivation with Bacillus sp. strain Oa_4, is homologous to members of the heat shock protein 70 (HSP70) molecular chaperone family. HSPs are involved in the folding of newly synthesized proteins and also play a crucial role in protecting plant cells from the damaging effects of heat stress ( Sung and Guy, 2003 ; Al-Whaibi, 2011 ). In the Arabidopsis genome, there are 14 HSC70 genes ( Cazale et al., 2009 ). In our study, three differentially expressed proteoforms of HSC70-1 were detected. The proteoforms have different pI value (5, 5.9, and 8.4, respectively) but similar molecular weights (119 kDa) (Supplementary Figure 2 ) and are arranged in a spot pattern characteristic to the protein post-translational modification that is likely linked to phosphorylation, Therefore, most likely, a single HSC70-1 gene is upregulated in apple cells by treatment with endophytic bacteria. Recently, HSPs have received considerable attention due to their function in innate plant immunity ( Park and Seo, 2015 ), while their role in plant–endophyte interactions remains vague. It has been shown that HSP70 accumulates during Phytophthora infestans -mediated hypersensitive responses and non-host resistance to Pseudomonas cichorii in tobacco ( Kanzaki et al., 2003 ). HSP70 silencing has been associated with increased susceptibility to Xanthomonas campestris pv. vesicatoria in pepper Capsicum annuum ( Kim and Hwang, 2015 ). HSC70-1, together with HSP90 and SGT1, regulates Arabidopsis immune responses and is involved in ABA signaling events ( Clement et al., 2011 ). Upregulation of HSPs has been shown in the fungal endophyte colonization of barley roots ( Larriba et al., 2015 ). Downregulation of HSPs induced by non-pathogenic E. coli in Arabidopsis has also been described ( Paungfoo-Lonhienne et al., 2010 ), but their role in interactions with endophytic bacteria has not yet been defined. In the present study, the results of String database query revealed a functional network of several proteins closely related to HSC70-1 that were upregulated after treatment with Bacillus sp. strain Oa_4 ( Figure 6 ). Chaperone-like protein CDC48B has been shown to function in the plant immune response ( Rosnoblet et al., 2017 ), and a protein related to ATP-dependent Clp protease ATP-binding subunit ClpC (CLPC1) has been shown to be involved in the import of proteins into the chloroplast in concert with stromal Hsp93 and Hsp70 chaperones ( Flores-Perez et al., 2015 ). Tubulin 8 (TUB8) and an isoform of actin 11 (ACT11) are components of cytoskeleton. Proteins His-HF, DAPF, and the product of the gene AT2G43090 are enzymes involve in amino acid synthesis. RidA is the enzyme crucial for synthesis of branched-chain amino acids in chloroplasts ( Niehaus et al., 2014 ) and was also shown to function as a N-chlorination-regulated, HSP90-like chaperone in bacteria ( Muller et al., 2014 ), but this function of RidA remains elusive in plants. The described network of functionally related proteins is involved in protein metabolism and cell development. Very prominent changes in protein abundance imply that the treatment with Bacillus sp. strain Oa_4 leads to reorganization of the cell development. This could be the same process that leads to the shoot development suppressing activity of this strain. Treatment with the Bacillus sp. strain Da_4 upregulated another proteoform of RidA, chaperone protein CPN60A, and ubiquitin-related RUB1 that are involved in a functional network related to protein expression and cellular development. This could indicate a plant cell response common for the mutualistic interaction with endophytes or at least Bacillus sp. bacteria. In addition, the Da_4 strain led to significant changes in a set of proteins mainly involved in plant defence response or oxidative stress regulation ( Figure 6 ). Glutathione-S-transferase (GSTU19) and O-methyl-transferase (OMT1) were upregulated ( Gong et al., 2005 ; Gall et al., 2015 ). PA2 ( Pandey et al., 2017 ), Ser protease inhibitor (AT2G38870), three proteoforms of Kunitz-type protease inhibitor (KTI2) ( Ledoigt et al., 2006 ; Habib and Fazili, 2007 ) were downregulated in plants bacterized with Da_4. In addition, not shown in Figure 6 , several proteoforms of the PR-10 and PR-5 family members, including Pru av 1-like protein and thaumatin 1A ( Rajam et al., 2007 ; Wu et al., 2016 ) were downregulated. The results imply that downregulation of stress- and defence-related proteins may be important in the less pronounced modulating effect of Da_4 strain on cellular redox balance. Taken together, our study has established that the endophytic bacteria of Bacillus and Pseudomonas spp. have a strain-specific capability to regulate apple shoot biomass accumulation and proliferation of auxiliary shoots in vitro . This suggests that endophytic biological interaction could help plant explants overcome abiotic stresses encountered in vitro and could be useful in the micropropagation of recalcitrant plant genotypes as an alternative to chemical treatment. We show here that molecular events implicated in the early formation stage of the plant host and endophytic bacteria interaction could reflect on the long-term outcome of the interaction and on plant phenotype. Modulation of the cellular redox balance in apple cells during the first hours of interaction could denote a bacterial strain-specific effect on apple shoot development in vitro , and the effect has a potential application as a biochemical marker useful for bacterial isolate screening. Further, the proteomic analysis revealed protein expression patterns specific to the strain-specific development of responses in the apple cells. This work provides hints about cell developmental reorganization and stress signaling processes involved in plant host and endophyte interactions under in vitro conditions, and it paves the way for further studies on the implicated mechanisms."
} | 6,068 |
38600179 | PMC11006893 | pmc | 6,011 | {
"abstract": "Stretchable neuromorphic optoelectronics present tantalizing opportunities for intelligent vision applications that necessitate high spatial resolution and multimodal interaction. Existing neuromorphic devices are either stretchable but not reconcilable with multifunctionality, or discrete but with low-end neurological function and limited flexibility. Herein, we propose a defect-tunable viscoelastic perovskite film that is assembled into strain-insensitive quasi-continuous microsphere morphologies for intrinsically stretchable neuromorphic vision-adaptive transistors. The resulting device achieves trichromatic photoadaptation and a rapid adaptive speed (<150 s) beyond human eyes (3 ~ 30 min) even under 100% mechanical strain. When acted as an artificial synapse, the device can operate at an ultra-low energy consumption (15 aJ) (far below the human brain of 1 ~ 10 fJ) with a high paired-pulse facilitation index of 270% (one of the best figures of merit in stretchable synaptic phototransistors). Furthermore, adaptive optical imaging is achieved by the strain-insensitive perovskite films, accelerating the implementation of next-generation neuromorphic vision systems.",
"introduction": "Introduction Advancing neuromorphic optoelectronic systems to mimic visual perception, adaptation, and imaging are essential for next-generation artificial intelligence equipment, such as medical auxiliary gadgets, augmented reality displays and bionic robots 1 – 4 . All-in-one neuromorphic vision device that can substantially improve the computing efficiency and elegance, has provoked intensive interest in computer vision and deep learning 5 , 6 . To achieve such high-level optoelectronics, innovations in photosensing materials, compact structural design and integration technology are indispensable 7 – 10 . Extensive efforts have been devoted to endowing photosensors with synaptic or adaptive behavior through developing two-dimensional materials with tunable energy levels 11 – 13 , engineering perovskite heterostructures 14 – 16 or building multilayer organic heterostructure 17 , 18 . However, the reported bioinspired optoelectronic devices and circuits generally exhibit monotonous functionality and severe performance degradation under mechanical deformations 19 , 20 , which cannot satisfy the multiple demands imposed by wearable and implantable electronics. Elastic neuromorphic vision sensors with excellent biocompatibility and mechanical compliance allow seamless attachment onto complex-shaped surfaces, naturally exhibiting high resolution and accuracy in mobile detection 21 , 22 . Recently, intrinsically stretchable neuromorphic optoelectronics have gained extensive attention owing to their free deformation and cross-scale modulus adaptability 23 . In particular, the intrinsically stretchable design undoubtedly enables more convenient fabrication, higher versatility and availability than complicated geometric engineering, becoming an inevitable componence for next-generation human-oriented applications. Notably, intrinsically stretchable organic phototransistors that have demonstrated low noise and high photoconductive gain, are capable of functional integration of vision and synapses 19 , 20 , 24 . Despite recent progress in adaptive phototransistors 11 – 13 , 17 , 18 , 25 , 26 , to date no case of the intrinsically stretchable adaptive sensor has been reported due to the stiff active materials and complicated architectures. Meanwhile, additionally introducing synaptic characteristics remains a daunting challenge for adaptive sensors with defect tunability, whether developing ionic conducting elastomer 27 – 29 , assembling multiple monofunctional devices 30 or utilizing bilayer semiconductors 31 . Toward high-efficiency processing and low-power multiplexing, innovations in broadband vision-inspired neuromorphic sensors are extremely desirable 32 . However, existing stretchable photosensitive materials typically have a narrow absorption range or limited mechanical ductility that no longer pertain 20 , 24 , 33 – 35 . Although efforts have been devoted to combine perovskites with elastomer to balance stretchability and photoconversion performance 36 , achieving strain-insensitive neuromorphic vision is still in its infancy. To tackle these challenges, exploiting excellent stretchable photosensitive materials and multifunction-integrated design is requisite for realizing intrinsically stretchable neuromorphic optoelectronic devices. Herein, we report an intrinsically stretchable neuromorphic vision-adaptive transistor (ISNVaT) made of the defect-tunable viscoelastic perovskite film. The perovskite dots are microspherically distributed in elastomers by surface energy-induced strategy, enabling the strain-insensitive quasi-continuous microsphere (QCM) morphology. Such viscoelastic perovskite films not only ensure intrinsic stretchability and retentive photosensitivity, but also provide tunable charge-trapping defects that can guide photoadaptation and synaptic behaviors. Employing QCM perovskite film and elastic semiconductor layer to construct the layer heterojunction, the obtained device presented trichromatic photoadaptation and high biaxial stretchability (up to 100%). For synapse simulation, the ISNVaT showed an ultra-low energy consumption of 15 aJ and a record high paired-pulse facilitation (PPF) index of 270% (that is the highest value among reported stretchable synaptic phototransistors). Furthermore, a fast adaptive speed down to 150 s was realized, promising high-resolution adaptive imaging that was comparable with human eyes. ISNVaT technology pushes forward the skin-like neuromorphic vision systems for the emerging applications including visual prosthetics, bioinspired robots and unmanned intelligence.",
"discussion": "Discussion Through excellent designs in the viscoelastic perovskite films and device engineering, we achieved a trichromatic neuromorphic vision-adaptive sensor based on intrinsically stretchable phototransistors. The described viscoelastic perovskite film with the quasi-continuous microsphere (QCM) morphology features intrinsic stretchability, retentive photosensitivity and defect tunability that can guide photoadaptation and synaptic behaviors. The resultant phototransistors exhibit an ultra-low energy consumption down to 15 aJ, a record high paired-pulse facilitation (PPF) index of 270% and a high biaxial stretchability up to 100%. Furthermore, a fast adaptation speed (< 150 s) was realized which was able to realize adaptive imaging beyond human eyes (3–30 min). Therefore, our ISNVaTs pave the way for visual prosthetics, bioinspired robots and unmanned intelligence."
} | 1,665 |
39211608 | PMC11350736 | pmc | 6,012 | {
"abstract": "Polyethylene terephthalate (PET) and glycerol are prevalent\nforms\nof plastic and biowaste, necessitating facile and effective strategies\nfor their upcycling treatment. Herein, we present an innovative one-pot\nreaction system for the concurrent depolymerization of PET plastics\nand the transesterification of glycerol into dimethyl terephthalate\n(DMT), a valuable feedstock in polymer manufacturing. This process\noccurs in the presence of methyl acetate (MA), a byproduct of the\nindustrial production of acetic acid. The upcycling of biowaste glycerol\ninto glycerol acetates renders them valuable additives for application\nin both the biofuel and chemical industries. This integrated reaction\nsystem enhances the conversion of glycerol to acetins compared with\nthe singular transesterification of glycerol. In this approach, cost-effective\ncatalysts, based on perovskite-structured CaMnO 3 , were\nemployed. The catalyst undergoes in situ reconstruction in the tandem\nPET/glycerol/MA system due to glycerolation between the metal oxides\nand glycerol/acetins. This process results in the formation of small\nmetal oxide nanoparticles confined in amorphous metal glycerolates,\nthereby enhancing the PET depolymerization efficiency. The optimized\ncoupled reaction system can achieve a product yield exceeding 70%\nfor glycerol acetates and 68% for PET monomers. This research introduces\na tandem pathway for the simultaneous upcycling of PET plastic waste\nand biowaste glycerol with minimal feedstock input and maximal reactant\nutilization efficiency, promising both economic advantages and positive\nenvironmental impacts.",
"conclusion": "Conclusions In this study, we introduce a novel approach\nfor concurrently upgrading\nthree waste feedstocks: PET plastic, biowaste glycerol, and industrial\nbyproduct MA, without requiring any additional feedstock. Through\nthe utilization of coupled tandem transesterification reactions, we\naccomplish two key subreactions: the acetylation of glycerol into\nglycerol acetates and the depolymerization of PET into DMT and HEMT,\nwithin a single catalytic process. Methanol, generated from the reaction\nbetween glycerol and MA, is subsequently consumed in situ by PET methanolysis,\noptimizing the feedstock efficiency. The selected catalyst, perovskite-structured\nCaMnO 3 , demonstrates remarkable versatility in catalyzing\nthe tandem reactions. Notably, the CaMnO 3 catalyst undergoes\nin situ activation during the first reaction, forming amorphous metal-glycerolate\nflake composites encapsulating CaMnO 3 nanoparticles. This\ngenerated composite serves as an effective catalyst for the subsequent\nstep of PET depolymerization into monomers. Furthermore, the catalyst\nexhibited remarkable reusability with a slightly enhanced performance\nobserved over successive reactions, attributed to the in situ generation\nof new catalytic sites. The Ca and Mn active sites exhibit a synergistic\neffect, facilitating PET depolymerization. This approach yields over\n70% glycerol acetate products and 68% PET monomers at 200 °C\nfor 7 h. This work pioneers a new avenue for the co-upcycling of plastic\nwaste and industrial biowaste with maximized feedstock utilization\nefficiency, promising economic benefits, and enhanced sustainability.\nIt is anticipated that these findings will inspire further research\nin this field, driving the development of highly efficient protocols\nfor transforming waste into valuable resources.",
"introduction": "Introduction With the escalating demand for plastic\nproduction and its widespread\ndaily use, urgently addressing global white pollution requires a focused\neffort on plastic waste recycling. 1 , 2 Despite the\nsubstantial annual production of plastic waste, only 9% undergoes\nsuccessful recycling. 3 Polyethylene terephthalate\n(PET), a common polyester plastic extensively employed in food packaging,\ndrink containers, and textiles, constitutes 13% of the total plastic\nproduction and is a key contributor to this waste stream. 4 Presently, PET waste is predominantly processed\nthrough mechanical recycling, involving melting and physical transformation,\nwithout inducing any chemical structure changes. However, this method\nunavoidably leads to deterioration of the properties and lower quality\nof PET products. In this context, chemical upcycling of PET waste\ninto depolymerized aromatic monomers or higher-value chemicals is\na more attractive recycling strategy. Various methods have been explored\nfor the valorization of PET waste, 5 , 6 including hydrogenolysis, 7 − 9 solvolysis, 10 , 11 pyrolysis, 12 photocatalytic 13 , 14 and electrochemical\nupcycling, 15 and hybrid processes. 16 − 19 However, most of these methods require additional inputs of valuable\nchemicals and energy resources, such as H 2 , alcohols, intensive\nheat, and electricity. Solvolysis is a method extensively studied\nfor addressing polyester\nplastic waste, with glycolysis and methanolysis standing out as the\ntwo representative approaches that have received the most attention.\nMethanolysis is a transesterification process to decompose PET into\ndimethyl terephthalate (DMT) and ethylene glycol (EG) by solvolysis\nwith methanol. 20 Compared to the glycolysis\nprocess, the final product DMT from methanolysis of PET is more convenient\nto purify due to its low solubility in water. DMT serves as a feedstock\nfor synthesizing polyester plastics, resins, films, and paint, or\nas a valuable additive in polymer manufacturing. 21 Its global price has fluctuated between 900 and 1300 US\ndollars per metric ton (USD/mt) over the past 5 years. 22 Current methodologies for PET methanolysis can\nbe categorized into two main approaches: supercritical reactions,\nendowed at high temperatures exceeding 250 °C and elevated pressures, 23 , 24 and catalytic methanolysis, which effectively mitigates the need\nfor extreme conditions by lowering both the reaction temperature and\npressure. 25 The commonly used catalysts\nfor PET methanolysis include homogeneous\ncatalysts such as metal acetates, 26 heterogeneous\ntransition-metal and alkali earth metal oxides, 27 and metal-free ionic liquids. 28 The homogeneous catalysts may cause product purification problems.\nCatalytic methanolysis was observed to proceed through the interaction\nbetween the transition-metal cation M n+ center and the\ncarbonyl groups within PET, resulting in the enhanced activation of\nthe O–H bond in methanol. 25 It has\nalso been reported for alkali earth metal catalysts; methanol is first\nactivated by M 2+ to form M–O–CH 3 which further reacts with the ester groups in PET. 27 However, in heterogeneous catalysis, the limited dispersion\nof catalytically active sites poses a challenge, impeding their efficient\ncontact with reactants, particularly polymers. There is a need to\naugment the effective interaction between catalytic sites and PET.\nIt is worth noting that while both alkali earth metal catalysts (e.g.,\nMgO and CaO) and transition-metal catalysts (e.g., Mn 3 O 4 , Fe 3 O 4 , and ZnO) have been investigated\nfor the alcoholysis of PET, 25 , 27 , 29 − 32 there has been limited exploration into enhancing catalytic performance\nby combining two or more metal oxides, leveraging the benefits of\na synergistic catalytic mechanism. Moreover, a high methanol/PET ratio\nis required to improve the overall methanolysis efficiency, which\nneeds a large amount of methanol loading. Given methanol’s\ncrucial role as a fuel source, solvent, and building block in the\ncontemporary chemical industry, obtaining methanol from less valued\nindustrial wastes or byproducts, such as biomass wastes, would be\na more favorable and sustainable approach. Glycerol, derived\nfrom biomass, undergoes extensive industrial\nproduction, constituting approximately 10% of the byproducts generated\nduring biodiesel manufacturing. 33 , 34 The burgeoning production\nof biodiesel has spurred a rise in glycerol output, resulting in oversaturation\nand a subsequent price decline. Presently, glycerol production has\nexceeded market capacity, rendering it a biowaste. Projections indicate\nthat biodiesel production will reach 60 billion liters by 2025, yielding\napproximately 6 billion liters of glycerol biowaste. 35 Consequently, urgent efforts are needed to devise efficient\nmethods for glycerol valorization before it becomes a pollutant. Glycerol’s\ninherent reactivity, attributed to its three hydroxyl groups, positions\nit as a highly promising precursor for the synthesis of diverse value-added\nproducts. 36 An attractive approach\nto upgrade glycerol is to convert it into\nglycerol esters by esterification or transesterification. 37 − 40 For instance, the acetylation of glycerol can produce glycerol acetates\n(acetins) as valued chemicals in diverse applications with increasing\nmarket demands. Acetins including monoacetin, diacetin, and triacetin\nare widely used for cosmetics, pharmaceuticals, food additives, and\nbiodiesel additives. 41 The average price\nof glycerol stands at 350 USD/mt, but its conversion into acetins\ncan elevate its value significantly, ranging from 1000 to 2000 USD/mt. 42 , 43 The conversion of glycerol into acetins can be achieved through\nthe transesterification of glycerol with methyl acetate (MA), a byproduct\nextensively generated in the manufacturing process of poly(vinyl alcohol)\n(PVA). 44 The worldwide annual production\nof PVA is over 1 Mt, leading to the generation of 1.68 Mt MA, suggesting\na promising alternative solution for the downstream utilization of\nglycerol. 45 The transesterification of\nglycerol and MA has been reported in several research papers using\nacid-modified silica, silica-supported yttrium, and lipase catalysts. 45 − 47 However, a comprehensive investigation into the reaction performance\nutilizing metallic catalysts is still pending. In addition to the\nthree glycerol acetate products, methanol is generated as a byproduct\nfrom the transesterification of glycerol and MA. This observation\nopens up the possibility of synergizing the transesterification processes\nof glycerol, MA, and PET plastics. The methanol produced in the reactions\nbetween glycerol and MA can potentially act as a reactant in the methanolysis\nof PET. In this work, a one-pot reaction system simultaneously\nmanaging\nPET plastic, MA, and glycerol via tandem transesterification is reported.\nIn this process, all of the reactants are wastes with low costs, and\nthe valorization occurred via the internal reactions among the three\nwastes with no additional feedstocks such as methanol needed. The\nindustrial biowaste glycerol is valorized into glycerol acetates (mono-,\ndi-, and triacetin) in the reaction with MA; meanwhile, the coproduct\nmethanol allows methanolysis for the depolymerization of PET plastics\ninto oligomers, then 2-hydroxyethyl methyl terephthalate (HEMT), and\nfinally the DMT monomers, as illustrated in Scheme 1 . Alkali earth-metal and transition-metal\ncatalysts were screened in this work, which presented dual activity\nin the two tandem transesterification reactions. Moreover, it was\nfound that the perovskite-type CaMnO 3 catalyst underwent\nin situ reconstruction during the tandem reactions within the PET/glycerol/MA\nsystem. This transformation occurred due to glycerolation between\nthe metal and glycerol/acetins, resulting in the formation of small\nmetal oxide nanoparticles confined within amorphous metal glycerolates.\nThis newly formed structure exhibits exceptional activity for the\ndepolymerization of PET to DMT monomers. This work demonstrates the\nco-upcycling of plastic waste and industrial biowaste to maximize\nthe feedstock utilization efficiency and attain higher sustainability. Scheme 1 Tandem Transesterification Reactions of MA, Glycerol, and PET Proposed\nin This Work",
"discussion": "Results and Discussion A transparent PET film with\nthe size of 5 mm × 5 mm was used\nin this work as shown in the image in Figure S1A . Diffraction peaks at 2θ = 23.2 and 26.0° with hkl values\nof (110) and (100), respectively, were identified for the PET crystalline\nstructure, as shown in Figure S1B . Since\nthe two proposed subreactions in this study involve transesterification,\nthe catalyst screening began with the catalysts that could potentially\nbe applied in both processes. We selected alkali earth metals Ca and\nMg, as well as transition metal Mn, as the starting materials to develop\na range of metal-based catalysts with diverse structures and compositions\nincluding Ca- and Mg-based catalysts [CaO, MgO, Ca, and Mg layered\ndouble hydroxide (LDH)] and Mn-based catalyst (MnO x ). Furthermore, a CaMnO 3 perovskite oxide was synthesized\nto investigate the performance of a dual-metal catalyst containing\nboth alkaline earth and transition metals. Prior to the coupled tandem\nreaction, the catalysts were first tested in two individual transesterification\nreactions, namely, glycerol acetylation and PET methanolysis. As depicted\nin Figure 1 A, the transesterification\nof glycerol and MA without PET (referred to as the GM reaction) was\ncarried out with a stoichiometric molar ratio of MA/glycerol = 3 at\n200 °C for 5 h. Compared to the catalyst-free test, all the adopted\ncatalysts exhibited remarkable activity, enhancing the product yield\nby at least 4-fold. There is minimal disparity in the activities of\nthe Ca, Mg, and Mn catalysts, suggesting that the GM reaction is readily\ncatalyzed by a diverse array of catalysts. The compositions of the\nglycerol acetate products are also similar among different catalysts.\nDue to the stoichiometric dosage of MA and glycerol, monoacetin constitutes\nthe largest portion of the products, followed by diacetin, with triacetin\nbeing the least abundant among the three. Previous studies have utilized\nan excess of MA relative to glycerol to enhance the yield of triacetin. 45 However, this approach represents a trade-off\nbetween the cost of feedstock and the desired product yield. Figure 1 Reaction behavior\nof different catalysts in (A) transesterification\nwith MA and glycerol (GM reaction); (B) methanolysis of PET with methanol;\n(C,D) tandem reaction of glycerol, MA, and PET (PGM reaction). Reaction\nconditions: (A) 10 mmol of glycerol, 30 mmol of MA, 2 mL of 1,4-dioxane,\n200 °C, 5 h, 1 atm argon. (B) 2 mL of 1,4-dioxane, 2 mL of methanol,\n0.3 g of PET film, 200 °C, 2 h, 1 atm argon. (C,D) 10 mmol of\nglycerol, 30 mmol of MA, 2 mL of 1,4-dioxane, 0.05 g of PET film,\n200 °C, 7 h, 1 atm argon. 1.8 mmol of the catalyst was loaded\naccording to its chemical formula. For combined catalysts, 3.6 mmol\nof MnO x and 1.8 mmol of MgO were loaded\nto achieve the same metal loading as MgMn 2 O 4 spinel oxides, and 1.8 mmol of MnO x and\n1.8 mmol of CaO were loaded to achieve the same metal loading as CaMnO 3 . The catalytic performance was then monitored in\nthe methanolysis\nreaction of PET with excess methanol loading at 200 °C for 2\nh. The reaction efficiency was assessed based on the yields of two\nmonomers resulting from PET depolymerization. These monomers, containing\na single aromatic ring in their molecules, can be purified and serve\nas feedstocks for the repolymerization process with EG to regenerate\nhigh-quality PET plastics. The yields of both DMT and HEMT are depicted\nin Figure 1 B. The degradation\nof PET barely occurred without a catalyst, while all the Ca- and Mg-containing\ncatalysts afforded a high yield of DMT, with the Ca-based catalyst\nshowing superior activity against the Mg-based ones to yield DMT close\nto 70% over CaAl-LDH. It is possibly due to the stronger basicity\nof Ca to activate the O–H bond of methanol. However, the formation\nof DMT and HEMT was scarcely observed over MnO x , indicating its inactivity for PET methanolysis. It is worth\nmentioning that CaMnO 3 exhibited lower activity compared\nto that of CaO and CaAl-LDH, despite containing an identical amount\nof Ca. This suggests that the composite catalyst formed by CaMnO 3 hindered the methanolysis efficiency of PET. The feasibility\nof applying the catalytic system into the designed\ntandem transesterification of glycerol, MA, and PET (referred to as\nPGM reaction) was explored afterward at 200 °C for 7 h without\nthe loading of methanol, and the product yields of glycerol acetate\nand DMT/HEMT are displayed in Figure 1 C,D, respectively. The distribution of the glycerol\nacetate products in PGM reactions is similar to that in GM reactions\nover different catalysts. The monoacetin yield fell into a range between\n45 and 60% for all the catalysts. MnO x shows slightly higher overall product yields compared to the Ca-\nand Mg-based catalysts, which contrasts with the GM reaction shown\nin Figure 1 A. This\nsuggests that MnO x boosted the performance\nof glycerol acetylation in the tandem reaction system. The PET depolymerization\nefficiency in this tandem reaction is reasonably lower than that in\ndirect methanolysis by methanol due to the lower methanol concentration\ngenerated from the reaction between MA and glycerol. Interestingly,\nMnO x achieved higher DMT and HEMT yields\nthan MgO, despite its inactivity in PET methanolysis, as shown in Figure 1 B. It is conceivable\nthat new catalytically active species were generated during the PGM\nprocess, rendering it capable of catalyzing the depolymerization of\nPET with the in situ-formed methanol. This phenomenon will be further\nexplored in the upcoming mechanism studies. The highest yields\nof DMT (32.8%) and HEMT (23.9%) were attained\nover CaMnO 3 , indicating that the combined presence of Ca\nand Mn in a single catalyst surpasses the performance of individual\nmetal oxides of Ca and Mn. For comparison, the combination of Mg and\nMn in the form of a single catalyst, namely, MgMn 2 O 4 spinel oxides, was also evaluated in the PGM tandem reaction.\nHowever, the PGM reaction performance of MgMn 2 O 4 spinel oxides is inferior to that of both MgO and MnO x , suggesting that the catalytic efficacy in PGM is\ninfluenced by the composition and structure of the mixed oxides, rather\nthan the simple accumulation of each metal component. For further\ncomparison, we tested the PGM reaction with both MnO x and CaO to achieve identical loading of Ca and Mn as CaMnO 3 . The product yield of this MnO x /CaO mixture is slightly higher than that of MnO x but still lower than that of CaMnO 3 . This indicates\nthat the CaMnO 3 catalyst outperforms each individual metal\ncomponent and its simple combination. Furthermore, the coexistence\nof both MnO x and MgO for PGM resulted\nin even poorer performance than the individual catalysts, indicating\na negative effect of this dual-catalyst configuration. We speculate\nthat this could possibly be attributed to the strong association of\nmethanol with MgO, which inhibited effective interaction with Mn-based\nactive sites to effectively react with PET since MgO and MnO x are separated particles. Therefore, CaMnO 3 is considered as the best catalyst in this tandem reaction system,\nwhere the activity does not simply accumulate from Ca and Mn. It was\nchosen as the representative catalyst for subsequent studies. To monitor the reaction process, track the evolution of product\nformation, and assess the impact of PET presence on the GM reaction\nefficiency, we utilized CaMnO 3 as the catalyst and conducted\nboth the GM and PGM reactions individually for a duration of 20 h.\nThe GM and PGM reactions were conducted under the same conditions\nexcept for the PET loading in PGM. As depicted in Figure 2 A, the monoacetin yield reached\n41% at 2.5 h in the GM reaction, which is identical to that observed\nin the PGM reaction. Subsequently, the product yield gradually increased\nover time, ultimately reaching 65% at the 20 h mark, with a cumulative\n78% yield attained for all three glycerol acetate products in the\nGM reaction. In the PGM reaction, the productivity surpassed that\nof the GM reaction for all three glycerol acetate products. Notably,\nfinal yields of 80 and 98% were achieved for monoacetin and total\nproducts, respectively, in the PGM reaction at 20 h. Hence, it can\nbe inferred that the glycerol acetylation process is enhanced in the\npresence of PET, potentially attributed to the consumption of in situ-produced\nmethanol, leading to a shift in the reaction equilibrium toward greater\nproduct formation. Figure 2 Comparison of the product evolution with reaction time\nfor the\nGM (without PET) and PGM (with PET) reactions. (A) Glycerol acetate\nyields in the GM and PGM reactions. (B) DMT/HEMT yields in the PGM\nreaction. Conditions: 10 mmol of glycerol, 30 mmol of MA, 2 mL of\n1,4-dioxane, 0.025 g of PET film, 200 °C, catalyst: 1.8 mmol\nof CaMnO 3 . The evolution of DMT and HEMT product yield in\nthe PGM reaction\nis presented in Figure 2 B. After 2.5 h of tandem reaction, 15.3% DMT and 13.1% HEMT were\nachieved. The DMT yield peaked at 7.5 h, reaching 34.7%, before gradually\ndeclining. Conversely, the HEMT yield continued to rise until 10 h\nof reaction and then reached a plateau of 34.5%. This phenomenon can\nbe understood through the following interpretations: initially, the\nmethanolysis of PET occurs at random points along the PET chain, leading\nto the production of oligomers. Subsequently, chain shortening ensues,\nresulting in the formation of HEMT which contains a single aromatic\nring with one side chain remaining to be released through the transesterification\nby methanol. Afterward, HEMT is ultimately transformed into DMT. However,\nafter 7.5 h of reaction time, the concentration of DMT reaches its\npeak, signifying the onset of enhanced and dominant repolymerization.\nDMT reacts with EG to form HEMT and continues to undergo further polymerization,\nleading to the formation of longer chains. Consequently, the decrease\nin DMT yield after 7.5 h can be attributed to this enhanced repolymerization\nprocess, while HEMT exhibits negligible change due to the coexistence\nof depolymerization and repolymerization. Similar repolymerization\nresults were also observed in the previously reported research on\nPET degradation by methanolysis and glycolysis. 27 , 48 The influence of the reaction conditions was also investigated\non the CaMnO 3 catalyst to gain a comprehensive understanding\nof the PGM reaction process. As shown in Figure S2A,B , the reaction temperature presented a significant influence\non the yield of products, especially the PET degradation. Both the\nyields of glycerol acetates and DMT/HEMT increased with elevated temperature.\nAt 170 °C, 36% monoacetin and 10% diacetin were gained, which\nwere gradually increased to 53 and 12%, respectively, at 200 °C.\nNevertheless, the DMT yield was only 0.5 and 5.7% at 170 and 180 °C,\nrespectively. At higher temperatures of 190 and 200 °C, the yield\nof DMT and HEMT was remarkably enhanced. This indicates that the glycerol\nacetylation subreaction requires lower activation energy than PET\ndepolymerization into DMT. The impact of the loading amount\nof CaMnO 3 on the PGM\nprocess was studied as summarized in Figure S2C,D . The increment of catalyst loading from 0.45 to 0.9 and 1.8 mmol\nresulted in a noticeable increase in glycerol acetates and PET monomers\nat a similar scale, attributed to the multiplication of catalytic\nsites dedicated to the reaction. However, upon further increasing\nthe CaMnO 3 dosage to 3.6 mmol, no significant increase\nin the glycerol acetate products was observed. Instead, there was\na slight decrease in DMT yield and a substantial increase in HEMT.\nThis could be explained by the equilibrium of the transesterification\ncaused by excess catalyst loading when the DMT started to convert\ninto HEMT by EG. The highest DMT + HEMT yields of 68% were observed\nat a CaMnO 3 dosage of 3.6 mmol. Notably, no PET residual\nwas observed in the product solution after the reaction, indicating\nthat all of the PET was depolymerized into soluble oligomers and monomers. Furthermore, the solvent 1,4-dioxane was removed or substituted\nwith other solvents to investigate its role in CaMnO 3 -catalyzed\nPGM. The results are illustrated in Figure S3 . Compared to PGM with 1,4-dioxane, the removal of the solvent led\nto a dramatic decrease in DMT yield from 32.8 to 7%. The solvent’s\nrole could be identified as facilitating the dissolution of short-chain\nPET molecules, allowing better contact with the catalyst and methanol\nto form DMT and HEMT. Additionally, three other solvents including\ntoluene, dichloromethane, and chloroform were also tested. The reaction\nefficiency over different solvents followed the sequence of 1,4-dioxane\n> chloroform > dichloromethane > toluene > solvent-free.\nThus, 1,4-dioxane\nemerged as the superior solvent in this PGM system. Meanwhile,\nthe composition of the three feedstocks was adjusted\nindividually to see the influence on the reaction yield. The results\nare summarized in Table S1 . When the MA\nloading was doubled from the reference of 10 mmol of glycerol, 30\nmmol of MA, and 0.05 g of PET film, a decrease in monoacetin, DMT,\nand HEMT yields by 16, 21, and 11% respectively, was observed. This\nreduction may be attributed to the decreased solubility of PET oligomers\nwith a higher portion of MA. Similarly, doubling the glycerol in the\nPGM system also resulted in reduced product yields. It is noted that\nthe high loading of glycerol could not fully dissolve into the reaction\nsolution, and the metal oxide catalyst exhibited a higher affinity\ntoward glycerol, becoming confined to the glycerol phase. This weakened\nthe interaction between the catalyst and the PET/methanol reactants.\nAdditionally, when the PET dose was reduced by 50%, there was no significant\nvariation in the product yield, indicating that the PET methanolysis\nequilibrium is not greatly shifted, as glycerol/MA is excessive compared\nto PET. The influence of the specific surface area (SSA) of\nthe CaMnO 3 catalyst was also studied in this work. In addition\nto the\nsol–gel preparation method, coprecipitation and hydrothermal\nmethods were employed to prepare CaMnO 3 catalysts with\nvarying SSA. The SSA of the catalysts follows the sequence hydrothermal\n> sol–gel > coprecipitation, as summarized in Table S2 . However, no clear correlation between\nthe catalytic\nperformance in the PGM reaction and the SSA was observed ( Figure S4 ). Specifically, the three catalysts\nproduced similar yields of acetins, while CaMnO 3 (sol–gel)\ndelivered a slightly higher DMT yield than those prepared by coprecipitation\nand hydrothermal methods. This indicates that the SSA of the catalyst\nis not the primary factor influencing the catalytic activity. The mass balance and carbon balance were calculated in the PGM\nreaction with CaMnO 3 . The calculation method is described\nin the Experimental Section . The total mass\nbalance of this system is calculated to be 96.0% ( Figure S5A ). The loss of mass balance was possibly due to\nthe reacted glycerol species with the catalyst and the mass loss during\nchemical transfer. The carbon balance, calculated from all the detected\ncomponents excluding the PET oligomers, was found to be 91.6% ( Figure S5B ). As previously mentioned, there\nmay be the generation of new catalytic\nsites over the MnO x catalyst during PGM\nreactions for PET depolymerization. This is evidenced by the shift\nobserved in MnO x from being inactive in\nPET methanolysis to becoming active in PGM. To elucidate the key component\ncontributing to the transition of MnO x , MnO x was pretreated in various environments.\nSubsequently, the MnO x catalysts were\ncollected by filtration, rinsed with 1,4-dioxane, and evaluated in\na PET/methanol reaction system. Five different treatment environments\nwere employed on MnO x at 200 °C for\n7 h: (1) 1,4-dioxane solvent only; (2) MA + solvent; (3) glycerol\n+ solvent; (4) GM reaction environment; (5) PGM reaction environment.\nThe results are presented in Figure 3 A. Figure 3 (A) PET methanolysis over MnO x catalysts\nafter different pretreatments. The treatments include (1) 1,4-dioxane\n(solvent); (2) MA + solvent (MA); (3) glycerol + solvent (Gly); (4)\nGM reaction environment; (5) PGM reaction environment. (B) FTIR spectra\nof fresh MnO x and MnO x after the GM reaction. For the PET methanolysis conducted on untreated\nMnO x and MnO x treated with\n(1) and (2), the overall DMT + HEMT yield was lower than 2%, indicating\nthat no active sites were generated. The DMT and HEMT yield was substantially\nimproved over the MnO x treated by (3),\n(4), and (5), suggesting that the interaction between MnO x and glycerol played a crucial part in the generation\nof new active sites. It has been reported that glycerol can form associations\nwith a variety of metals due to its abundant hydroxyl groups, leading\nto the formation of metal glycerolates ( M -glycerolates). 49 These compounds significantly enhance the exposure\nof metal sites, making them accessible for catalytic reactions. Transition\nmetals such as Zn, Mn, Fe, Co, and Ni are known to readily associate\nwith glycerol in this manner. 50 , 51 Moreover, recent research\nhas unveiled a significant discovery regarding PET glycolysis, revealing\nthat the Zn catalyst has the capability to engage with EG, leading\nto the formation of Zn-glycolate species. 48 These species have been proven to be efficient catalytic agents\nin the degradation of PET via glycolysis. Drawing inspiration from\nthis observation, we hypothesize that during the GM reaction, glycerol\ncan similarly interact with MnO x to produce\nMn-glycerolate species, which exhibit catalytic activity in the methanolysis\nof PET with enhanced accessibility to PET flakes. To verify\nthe successful glycerolation of MnO x ,\nthe surface chemical bonds of MnO x after\nGM treatment were analyzed by using Fourier transform infrared\n(FTIR) spectra within the wavelength range of 500–4000 cm –1 . As depicted in Figure 3 B, no distinct peaks were detected on fresh\nMnO x , while signals corresponding to alkyl\nchains and OH group vibrations appeared in the spectral regions at\n2930 and 3380 cm –1 , respectively. Peaks at 1720,\n1560, and 1420 cm –1 are assigned to C=O,\nC–O–C, and C–OH, respectively, 52 indicating the association of the MnO x catalyst with both glycerol and glycerol acetates. X-ray diffraction\n(XRD) analysis of fresh MnO x revealed\nstrong patterns of Mn 2 O 3 and MnO 2 ( Figure S6 ). Following GM treatment,\nthe peaks for Mn 2 O 3 and MnO 2 nearly\nvanished, consistent with the existing literature, suggesting the\ntransformation of the crystalline structure of metal-based materials\ninto an amorphous state due to the formation of M -glycerolates. 53 Specifically, the XRD\ndata in Figure S6 indicate that after the\nreaction, the Mn 2 O 3 signals became higher and\nsharper than the MnO 2 signals. To gain a better understanding\nof this phenomenon, XPS Mn 2p spectra of MnO x before and after the reaction were analyzed. The results in Figure S7 suggested that the ratio of Mn 3+ :Mn 4+ for reacted MnO x (1.47) was slightly lower than that of fresh MnO x (1.50). As the decrease and broadening of the XRD signal are\ndue to the generation of amorphous Mn-glycerolate species, we conjectured\nthat this could possibly be because the Mn 4+ cations with\nhigher valence state are more active than Mn 3+ in associating\nwith the O atoms to form Mn-glycerolate. Consequently, Mn 4+ became amorphous on the surface of the catalyst, leading to a broadened\nXRD signal but a higher XPS proportion than Mn 3+ . Furthermore, we performed GM treatment for CaO and CaMnO 3 , with the FTIR spectrum demonstrating analogous peaks of the fresh\nand GM-treated samples as shown in Figure 3 B, suggesting that CaO and CaMnO 3 can also generate M -glycerolate species ( Figure S8 ). Notably, Figure 3 A illustrates that the yields of DMT and\nHEMT from GM-treated MnO x surpassed those\nfrom glycerol alone, leading us to infer that under GM and PGM reaction\nconditions, Mn interacts with both glycerol and glycerol acetates\nto form mixed Mn-glycerolate species that serve as the boosted active\nsites for PET depolymerization. The reusability of CaMnO 3 in PGM reactions was tested\nin four cycling runs. The characterization of the filtered catalyst\nalso shed light on the new active site generation over CaMnO 3 during the PGM reaction. Following the PGM reaction, the initial\nblack CaMnO 3 powder transitioned to a brown hue, as depicted\nin Figure S6A,B . To investigate the structure\nevolution of the catalyst during the cycling runs, both the fresh\nand the spent catalysts were characterized by XRD, TG analysis, X-ray\nphotoelectron spectroscopy (XPS), and scanning electron microscopy\n(SEM) imaging. The XRD spectrum of the fresh catalyst displayed typical\npatterns of CaMnO 3 structures indexed to the orthorhombic\nperovskite, in accordance with the standard card, 54 − 56 while after\nthe PGM reaction, these signals nearly vanished ( Figure 4 A), aligning with the results\nobserved for MnO x ( Figure S6 ), thus confirming the formation of amorphous M -glycerolates on CaMnO 3 . The mass of the filtered\ncatalyst after the reaction was higher than that of the loaded fresh\ncatalyst due to the generated M -glycerolates. TGA\nconducted in air to determine the formed organic species on the catalyst\nrevealed continuous weight loss for CaMnO 3 after the first\nrun until reaching 735 °C, with a total weight loss of 52.1%,\ncontrasting the absence of weight loss on fresh CaMnO 3 ( Figure 4 B). After the second\nrun, the CaMnO 3 catalyst demonstrated an even greater weight\nloss, reaching 58.1%. This observation indicates that the continuous\nformation of metal glycerolates occurred during the tandem reaction.\nFor the cycling experiment, the used catalyst underwent filtration,\nwashing with 1,4-dioxane, and drying for subsequent use. Since the\nweight of the catalyst increased after the reaction, the mass loading\nof the catalyst for the next cycling run was recalculated based on\nthe TGA results to ensure consistent loading of metal content with\nthe former cycling run. As illustrated in Figure 4 C,D, the four cycling runs exhibited no discernible\nloss of activity in producing glycerol acetates and PET monomers.\nSpecifically, the production of glycerol acetates remained almost\nunchanged throughout the cycles. During the PET depolymerization,\nthe production of monomers exhibited a slight increase in the third\nand fourth runs, potentially attributed to the in situ generation\nof catalytically active species. This underscores the excellent reusability\nof the CaMnO 3 catalyst in facilitating this tandem reaction.\nWe also conducted inductively coupled plasma-optical emission spectrometry\n(ICP-OES) to detect the metal leaching within the filtered reaction\nsolution after the catalytic reaction. Only a trace amount of the\nMn cation was detected in the product solution from the fresh catalyst.\nThis trace amount of Mn leaching possibly originated from the surface\ndefect sites on the catalyst generated during the catalyst preparation.\nConsidering the excellent catalytic performance during the cycling\nruns, it can be inferred that there was no loss of active-metal sites\nduring the reaction. Figure 4 (A,B) Reusability test of CaMnO 3 in the PGM\nreaction\nfor 4 runs. Conditions: 10 mmol of glycerol, 30 mmol of MA, 2 mL of\n1,4-dioxane, 0.05 g of PET film, 200 °C, 7 h, catalyst loading:\n1.8 mmol of CaMnO 3 . (C) XRD patterns of fresh and used\nCaMnO 3 . (D) TGA of fresh and used CaMnO 3 . SEM images of fresh CaMnO 3 and CaMnO 3 after\nthe first and third runs depict morphological changes in the catalyst\nduring the cycling test ( Figure 5 A–C). Compared to the fresh CaMnO 3 , the surface of catalyst particles exhibited slight roughening after\nthe first run due to the presence of glycerolates ( Figure 5 A,B). Following the third run,\nmetal glycerolates accumulated, forming small flakes on the catalyst\n( Figure 5 C). The high-resolution\ntransmission electron microscopy (HR-TEM) images of fresh CaMnO 3 revealed a uniform lattice structure of the CaMnO 3 phase ( Figure 5 D,E).\nScanning transmission electron microscopy (STEM) and energy-dispersive\nX-ray spectroscopy (EDS) mapping in Figure 5 F further corroborated the formation of the\nCaMnO 3 structure, demonstrating an even dispersion of Ca,\nMn, and O throughout the catalyst. The TEM image of the filtered catalyst\nafter the PGM reaction ( Figure 5 G) exhibited the formation of metal-glycerolate flakes due\nto the reaction between CaMnO 3 and glycerol. The HR-TEM\nimages in Figure 5 H,I\nreveal that the filtered catalyst exhibits an amorphous structure\nwith crystal nanoparticles dispersed randomly, each averaging several\nnanometers in size. STEM and EDS mappings in Figure 5 J indicate the widespread dispersion of both\nCa and Mn throughout the flakes, confirming the formation of metal-glycerolate\nspecies. This observation suggests that during the tandem reaction,\nglycerol and acetins reactively decompose the CaMnO 3 catalyst,\nresulting in the formation of amorphous metal-glycerolate flakes that\nencapsulate small CaMnO 3 nanoparticles, in accordance with\nthe XRD results. The XPS O 1s spectra were deconvoluted into three\nmain species, namely, Mn–O–C (533.2 eV), lattice oxygen\n(530.7 eV), and adsorbed oxygen (527.5 eV). Deconvolution of the XPS\nO 1s spectra unveiled a significant decrease in lattice oxygen on\nthe fresh CaMnO 3 following the tandem reaction. This decline\nwas accompanied by an increase in M–O–C species ( Figure 6 ), providing further\nvalidation for the transformation of crystalline metal oxides into\namorphous metal glycerolates. Such a transformation effectively enhances\nthe activity of Mn species in facilitating the methanolysis of PET,\nultimately yielding monomers. Figure 5 (A–C) SEM images of CaMnO 3 : (A) fresh sample.\n(B,C) filtered samples after the first and third run of the PGM reaction,\nrespectively. HR-TEM images (D,E) and STEM and EDS mapping (F) of\nthe fresh CaMnO 3 catalyst. TEM (G), HR-TEM (H–I),\nand STEM and EDS mapping (J) of the filtered CaMnO 3 catalyst\nafter the PGM reaction. Figure 6 Deconvolution of O 1s spectra of XPS of fresh CaMnO 3 and CaMnO 3 after the PGM reaction. To directly confirm the formation of M -glycerolates\non CaMnO 3 after the PGM reaction, the reacted CaMnO 3 underwent thorough washing with 1,4-dioxane followed by drying.\nSubsequently, hydrolysis with water was carried out to extract the\nglycerol and glycerol acetates from the M -glycerolates\non the catalyst. This hydrolysis process took place in water at 90\n°C for 48 h. The resulting precipitate was filtered, and the\nsolution underwent analysis using gas chromatography–mass spectrometry\n(GC–MS). Figure S9C–G illustrates\nthe GC spectrum, where a broad peak corresponding to glycerol was\nidentified. Additionally, a smaller peak, attributed to monoacetin,\nwas observed following the glycerol peak. No peaks indicative of diacetin\nor triacetin were detected in the GC spectra, potentially due to concentrations\nfalling below the GC–MS detection limit. These findings offer\nrobust evidence supporting the formation of M -glycerolates\non CaMnO 3 during the PGM reaction, facilitated by the interaction\nbetween metal oxides and glycerol/glycerol acetates. Based on\nthe discussion above, the possible mechanism of the PGM\nreaction process by CaMnO 3 is illustrated in Scheme 2 . First, perovskite-type CaMnO 3 serves as a catalyst for the transesterification of glycerol\nand MA, yielding glycerol acetates and methanol. Concurrently, both\nglycerol and acetins can bind with Ca and Mn to create catalytic composites\ncomposed of amorphous metal-glycerolate encapsulating small CaMnO 3 nanoparticles. The presence of Mn-glycerolate may enhance\nthe interaction between Mn and PET/methanol due to the increased affinity.\nThe lone electron pair in the oxygen of the C=O bond from PET\nis attracted by the Mn 4+ ion in Mn-glycerolate, facilitating\nester bond cleavage. Adjacent to Mn, the Ca atom activates the methanol\nmolecule through O–H bond cleavage, and the lattice oxygen\nO – abstracts the H from the hydroxyl group of methanol\nto form CH 3 O – . This species subsequently\nassociates with the carbon in the ester bonds of PET, leading to chain\nbreakdown into oligomers. Meanwhile, the Ca 2+ cations can\nprotonate the C=O groups in ester groups of PET, rendering\nthese ester groups more electrophilic and, thus, more susceptible\nto nucleophilic attack by methanol. This process results in the production\nof HEMT, which further converts into the DMT monomer. Scheme 2 Proposed\nMechanism of M -Glycerolate Species Generation\nand Catalytic PEM Reaction Finally, in terms of the real application of\nthis tandem reaction\nsystem, a crucial challenge is the separation and purification of\nthe products as complex mixtures will be generated. PET monomers can\nbe separated via crystallization by the addition of another solvent.\nMoreover, the nonvolatile glycerol derivatives can be separated from\nthe solvent and volatile products by distillation. The unreacted feedstocks\nmust be collected for reuse. Our work introduces a promising route\nfor waste co-upcycling, though further investigation will be needed\nfor practical application."
} | 10,277 |
34417171 | PMC8378814 | pmc | 6,013 | {
"abstract": "Self-contained soft electrofluidic actuators display excellent performance in portability, rapid response, and actuation.",
"introduction": "INTRODUCTION In contrast to conventional rigid robots, soft robots ( 1 – 8 ) made from flexible materials offer great advantages in adapting to complex surroundings, performing autonomous tasks, and mimicking the motions and functions of biological systems, which have the potential to be used for more compliant and safer devices that operate close to or even in human. A lot of impressive and attractive work about soft robots have been reported. For example, Shepherd et al. ( 4 ) introduced a pneumatically actuated quadrupedal robot that could move in narrow spaces. Mao et al. ( 5 ) fabricated a flower-shaped robot with five soft electromagnetic actuator petals, of which each petal can be programmed and operate very fast. Tang et al. ( 6 ) developed a high-speed cheetah-like galloping crawlers with ultrafast locomotion speeds of 2.68 body length per second by skillfully leveraging elastic instabilities for improving performances of soft pneumatic robots. As the core component of soft robots, there exist persistent challenges for developing high-performance soft actuators that can achieve several indispensable properties, including controllability, portability, durability, safety, versatility, rapid response, and excellent actuation. Different from rigid actuators, soft actuators ( 5 , 9 – 16 ) need to deform themselves to achieve the motions or operations of soft robots. On the basis of different actuation and deformation mechanisms, a wide variety of soft actuators have been reported. Among these, traditional soft fluidic (pneumatic or hydraulic) actuators ( 6 , 15 ) are most prevalent because of their simple and versatile designs. Nevertheless, the requirement of external bulky, rigid compressors or pumps for supplying for pressurized fluids limits their portability ( 17 – 19 ). Besides, many portable soft actuators ( 9 – 10 , 13 – 14 ) driven by external stimulus, such as thermal energy, magnetism, humidity, and light, have also been developed. Although they have some own particular merits in some areas, some remarkable shortcomings, such as poor controllability and slow response speed, prohibit their widespread applications. For example, shape memory alloy actuators can provide large actuation force and high-power density ( 20 ), but the lack of controllability limits their applications because of poor control of thermal energy ( 21 ). Compared with other stimuli-responsive soft actuators, the development of advanced electrically responsive soft actuators has attracted considerable interests because the electricity, as the most commonly used energy, has excellent controllability and is easily compatible or seamlessly integrated with existing electric-driven products in our life and industry, providing the best platform for popularizing these soft actuators. Dielectric elastomer (DE) actuators ( 22 – 24 ) can achieve actuation of elastomer films through electrostatic extrusion effect between equal-area positive and negative electrode pairs, which stand out among various electrically responsive actuators for their rapid and controllable actuation with large amplitudes. Nevertheless, the dielectric breakdown seems to be a challenge for their reliable applications ( 25 – 26 ). Recently, a class of HASEL (hydraulically amplified self-healing electrostatic) actuators ( 11 ) were developed by replacing the solid dielectric inside the DE actuators with liquid dielectric, allowing for self-healing after dielectric breakdown. In addition to electrostatic extrusion mechanism, electroconjugate fluid (ECF) is a kind of electrically responsive functional fluid that can produce strong jet under a nonuniform electric field. The ECF jet pumps have been used to power soft robotic hands ( 27 ), inchworms ( 28 ), and muscle cells ( 29 ). The ECF jetting method provides a good way to address the poor portability problem of traditional soft fluidic actuators, while retaining the advantages of their simple and versatile designs. Current applications of ECF to soft actuators mainly focus on fluidic actuators driven by the separated ECF pumps, limiting the response speed of fluidic actuators. The more compact and simpler architecture of soft fluidic actuators that highly integrate ECF pumps, tubes, liquid reservoirs, and actuators still needs to be explored to further improve the response speeds of fluidic actuators. Here, we introduce a class of self-contained soft electrofluidic actuators (SEFAs), which mainly use electrically responsive fluids to drive the external elastomer pouch deformation. SEFAs have a simple and compact architecture with a rod positive electrode immersed in dielectric liquid and the ground electrodes coating the pouch surface, thereby guaranteeing that the actuators can be fully enclosed in a closed electrode region to generate ECF jet of dielectric liquid. Meanwhile, a method of dissociating hydrogel into dielectric liquid is proposed to further enhance the ECF effect, improving the actuation performances of SEFAs. SEFAs have several distinctive qualities: (i) SEFAs that highly integrate pumps, tubes, liquid reservoirs, and actuators not only exhibit good portability but also achieve excellent response to electrical signals; (ii) SEFAs have good safety because the high-voltage positive electrodes are encapsulated in dielectric liquid and the positive electrodes also do not need to deform during the actuation process; (iii) SEFAs are versatile, which can easily achieve different actuation modes, such as linear actuation and bending actuation, by just adjusting the stiffness or thickness of the external membrane shell; (iv) SEFAs have high environment adaptability, which enables them to operate in both water and air; (v) SEFAs are electrically powered, which grants them good compatibility with existing electric-driven products; (vii) SEFAs can be easily manufactured using basic fabrication techniques and widely available materials; thus, it is easy to be popularized. Successful applications of SEFAs in an artificial muscle stretching a joint and a soft bionic ray swimming in a tank demonstrate their excellent performances, illustrating their great potential to be popularized in soft robotics.",
"discussion": "DISCUSSION Here, we have explored a class of self-contained SEFAs, which are only manufactured by using basic fabrication techniques and widely available materials but exhibit excellent performances in safety, reliability, controllability, durability, versatility, rapid response, and excellent actuation. Meanwhile, the improvement method of dielectric liquid is proposed to enhance ECF jet in a closed elastomer, thereby improving the actuation performance of SEFAs. In addition, we have also developed HVPCs to power and control the SEFAs, offering a platform for research and development of the SEFAs as well as other electrostatic actuators. Artificial muscle stretching a joint, a compliant gripper catching a live goldfish, and a soft bionic ray swimming in a tank illustrate the wide potential of SEFAs for next-generation soft robotics. Relative to existing fluidic actuators ( 10 , 15 – 19 , 27 – 29 ), including soft fluidic actuators driven by separated ECF jet pumps or traditional rigid pumps, SEFAs that highly integrate pumps, tubes, liquid reservoirs, and actuators not only exhibit better portability but also show excellent response to electrical signals. Several detailed actuation performances of the SEFAs and some existing soft actuators, including strain rate, power, and energy density, are indicated in table S2. SEFAs are expected to consume more electrical power than DE actuators and HASEL actuators because of the viscous loss of fluid flow. Actuation performance of SEFAs could be further improved by adjusting the hardness and thickness of the elastomer membrane or the characteristics of the dielectric liquid. Overall, SEFAs strike a balance among several indispensable properties for human-robot interaction devices, including safety, controllability, rapid response, and excellent actuation. Although SEFAs present several promising features, there remain some challenges to resolve current limitations of SEFAs. An existing hurdle is that the dielectric liquid driven by high voltage is used in the current SEFAs, and future studies need to reduce the operating voltage through the use of alternate fluids that could be driven by low voltage or alternate materials of pouch. Meanwhile, there is a potential risk that the high-voltage electrode will be attracted to contact the solid pouch so as to cause the dielectric breakdown of the pouch if the actuator is strained too much. Therefore, it is better to find some effective methods to protect the membranes that are close to the positive electrode. Tungsten electrodes and hydrogel electrodes are used in this work. The problems of physical piercing and the mismatch between tungsten electrodes and soft surrounding membranes exist, while these problems could be addressed by the use of hydrogel electrodes. Therefore, other suitable soft conductive materials, such as conductive silicone, could also be used to avoid these problems. In addition, SEFAs consume more electrical power than other electrostatic actuators such as DE actuators or HASEL actuators because of energy loss of fluid flow. In this study, the electrically responsive fluids in a closed elastomer are used to develop SEFAs, but other ways of stimuli-responsive fluids, such as magnetically responsive fluids and photo-responsive fluids, might also be explored to achieve this target. Although the preliminary exploration of the wireless power transfer technology is conducted to power the SEFA, a one-to-many and long-distance all-round wireless power supply method should be explored in the future."
} | 2,475 |
21245846 | PMC3049413 | pmc | 6,014 | {
"abstract": "Fermentation of plant biomass by microbes like Clostridium phytofermentans recycles carbon globally and can make biofuels from inedible feedstocks. We analyzed C. phytofermentans fermenting cellulosic substrates by integrating quantitative mass spectrometry of more than 2500 proteins with measurements of growth, enzyme activities, fermentation products, and electron microscopy. Absolute protein concentrations were estimated using Absolute Protein EXpression (APEX); relative changes between treatments were quantified with chemical stable isotope labeling by reductive dimethylation (ReDi). We identified the different combinations of carbohydratases used to degrade cellulose and hemicellulose, many of which were secreted based on quantification of supernatant proteins, as well as the repertoires of glycolytic enzymes and alcohol dehydrogenases (ADHs) enabling ethanol production at near maximal yields. Growth on cellulose also resulted in diverse changes such as increased expression of tryptophan synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. This study gives a systems-level understanding of how this microbe ferments biomass and provides a rational, empirical basis to identify engineering targets for industrial cellulosic fermentation.",
"introduction": "Introduction Cellulosic biomass is the world's most abundant biological energy source ( Leschine, 1995 ). Recycling this vast carbon sink by cellulolytic microbes is one of the largest material flows in the global carbon cycle ( Falkowski et al, 2000 ). Microbes could industrially convert over 1.3 billion metric tons of cellulosic biomass to fuels and chemicals per year in North America ( Perlack et al, 2005 ), which could sustainably provide enough ethanol for 65% of US ground transportation fuel at current levels ( Somerville, 2006 ). However, plant biomass is composed primarily of high-molecular weight polysaccharides in a quasicrystalline structure, making the deconstruction of biomass a key challenge to developing cellulosic biofuels ( Houghton et al, 2006 ). Consolidated bioprocessing ( Lynd et al, 2002 ) is a promising strategy to overcome biomass recalcitrance by using microbes such as Clostridium phytofermentans that secrete enzymes to both depolymerize biomass and then ferment the resulting hexose and pentose sugars to a biofuel such as ethanol. C. phytofermentans is a mesophile from forest soil that ferments both of the main components of plant biomass, cellulose and hemicellulose, to ethanol and hydrogen ( Warnick et al, 2002 ). As a group 14 clostridium, this microbe is phylogenetically distant from well-studied cellulolytic clostridia. The C. phytofermentans genome encodes 161 carbohydrate-active enzymes (CAZy) including 108 glycoside hydrolases spread across 39 families ( Cantarel et al, 2009 ), highlighting the elaborate set of enzymes needed to breakdown different biomass types. Hydrolases in most clostridia have dockerin domains to bind a scaffolding protein on the cell exterior forming a multienzyme cellulosome. C. phytofermentans lacks scaffolding and dockerin domains, suggesting that cellulolytic enzymes are either freely secreted or are anchored to the cell in a novel, cellulosome-independent manner. Faced with the complexity of metabolizing biomass, systems-level strategies are needed to identify hydrolases and metabolic enzymes to engineer microbes for improved cellulosic bioconversion. We demonstrate such a strategy ( Figure 1 ) in C. phytofermentans by integrating analyses of growth, fermentation, enzyme activities, and electron microscopy with quantitative mass spectrometry-based proteomics of more than 2500 proteins. Protein concentrations were estimated by machine learning-supported spectral counting (Absolute Protein EXpression, APEX) ( Lu et al, 2007 ). Protein levels on hemicellulose and cellulose relative to glucose were determined using reductive methylation ( Hsu et al, 2003 ; Boersema et al, 2009 ), here called reductive dimethylation (ReDi) labeling, to chemically incorporate hydrogen or deuterium isotopes at lysines and N-terminal amines of tryptic peptides. We show that ReDi labeling gives accurate, low-cost quantification of a microbial proteome and can be used to discern extracellular proteins. C. phytofermentans expressed more than 100 CAZy and adapted their stoichiometries to each cellulosic substrate. Cellulosic fermentation entailed additional changes such as increased tryptophan and nicotinamide synthesis, use of alternative glycolytic enzymes, and adhesion to the substrate. We describe how these data provide a blueprint showing promising genetic targets to engineer microbes for more efficient conversion of biomass to fuels and biomaterials.",
"discussion": "Discussion Cellulolytic microbes such as C. phytofermentans reveal novel strategies for developing microbes to overcome the recalcitrance of cellulosic feedstocks, currently the main barrier to cellulosic biofuels ( Houghton et al, 2006 ). We analyzed C. phytofermentans growth and fermentation of different cellulosic polymers ( Figure 2 ), comprehensively quantified proteome changes that enabled fermentation of each substrate, and distilled the data into a model of cellulosic fermentation ( Figure 7 ) showing key enzymes that can be engineered in this bacteria and other hosts to potentially optimize cellulosic biofuels. Growth on biomass involves secretion of numerous CAZy as well as proteins for motility, binding of extracellular solutes, proteolysis, and formation of a proteinaceous surface layer ( Figure 4D ). Oligosaccharides were uptaken before breakdown by intracellular CAZy, enabling more efficient sugar transport, conserving energy by phosphorolytic cleavage, and ensuring the sugar monomers were not available to competing microbes. Sugars were catabolized using an EMP glycolysis incorporating reversible, PPi-dependent glycolytic enzymes and fermented using pyruvate ferredoxin oxidoreductase and multiple ADHs. Growth on cellulose also resulted in broad metabolic changes such as increased tryptophan and nicotinamide synthesis and repression of fatty acid synthesis ( Figure 6 ), which suggest ways to optimize cellulosic fermentation by supplementing growth media or genetic modification. Cellulosic bioconversion can be accelerated by expression of cellulolytic enzymes in their native hosts as well as in model organisms such as yeast ( Den Haan et al, 2007 ; Tsai et al, 2009 ) and E. coli ( Steen et al, 2010 ). C. phytofermentans cellulolytic enzymes may be particularly well suited to enable cellulosic bioconversion by heterologous expression. Unlike other clostridia that package cellulolytic enzymes into a cellulosome, C. phytofermentans freely secretes these enzymes such that they can be functionally expressed without relying on a cellulosomal scaffold. However, considering the C. phytofermentans genome encodes 161 CAZy, it would take 12 880 experiments to find the most effective enzyme pair for any particular substrate. High throughput methods such as quantitative proteomics are thus needed to systematically quantify the enzymes used by cellulolytic microbes to metabolize different biomass types. In this study, we build upon proteomic studies of cellulosomes in other clostridia ( Gold and Martin, 2007 ; Raman et al, 2009 ; Blouzard et al, 2010 ), to show how C. phytofermentans alters the stoichiometries of more than 100 CAZy ( Figure 5 ) as well as diverse metabolic processes ( Figure 6 ) when fermenting cellulosic substrates. By identifying the most highly upregulated and the secreted enzymes, we can prioritize targets for more efficient bioconversion of different types of biomass ( Ito et al, 2010 ). This approach is supported in that Cphy3367, which is the second most highly upregulated CAZy on cellulose and was secreted into the supernatant ( Figure 5B ), has recently been shown to be essential for cellulolysis ( Tolonen et al, 2009 ) and to solubilize cellulose in vitro ( Zhang et al, 2010a ). In this study, we focused on chemically defined substrates. In the future, we will apply these methods to untreated plant biomass composed of diverse polysaccharides and non-fermentable, soluble components. In addition to cellulolytic enzymes, cellulosic biofuels can be optimized by more efficient fermentation. C. phytofermentans ferments cellulose to ethanol with high specificity ( Figure 2F ), supporting that accelerating cellulose deconstruction will give larger gains than streamlining end products. This high fermentation efficiency also suggests heterologous expression of the alternative glycolytic enzymes and multiple ADHs from C. phytofermentans ( Figure 7 ) could improve ethanol yields in other microbes. C. phytofermentans grows rapidly on hemicellulose ( Figure 2B ), but produces more acetate relative to ethanol ( Figure 2E ). Faster growth on hemicellulose than on glucose or xylose may result from faster uptake of xylosaccharides by high-affinity transporters, energy savings because of transport of oligosaccharides relative to sugar monomers ( Muir et al, 1985 ), or the presence of an unidentified xylosaccharide phosphorylase. Higher relative acetate produced on hemicellulose than on cellulose is also supported by higher expression of acetate kinase, which could be inactivated to improve ethanol yields on hemicellulose-rich substrates. Additionally, acetate accumulation in hemicellulose cultures may partially result from cleavage of acetyl side chains from xylose subunits, which are common to hardwood xylans such as the birchwood used here ( Shallom and Shoham, 2003 ). Proteome-wide expression changes by ReDi proteomics are consistent with mRNA measurements by qRT–PCR ( Figure 3F ), but are advantageous in directly quantifying proteins and allowing discrimination of secreted and cellular enzymes. ReDi labeling is advantageous to stable isotope labeling by amino acids in cell culture (SILAC) ( Ong and Mann, 2006 ), a widely used alternative approach for stable isotope incorporation, in not requiring strains with specific amino acid auxotrophies or optimization of growth on synthetic medium. As ReDi uses inexpensive, highly quantitative chemistry and a small mass tag, it is easily incorporated into routine proteomics protocols ( Boersema et al, 2009 ). ReDi proteomics will be a key part of research to enable cellulosic biofuels and can be applied to many other research areas as well."
} | 2,619 |
20307315 | PMC2852388 | pmc | 6,015 | {
"abstract": "Background Microorganisms possess diverse metabolic capabilities that can potentially be leveraged for efficient production of biofuels. Clostridium thermocellum (ATCC 27405) is a thermophilic anaerobe that is both cellulolytic and ethanologenic, meaning that it can directly use the plant sugar, cellulose, and biochemically convert it to ethanol. A major challenge in using microorganisms for chemical production is the need to modify the organism to increase production efficiency. The process of properly engineering an organism is typically arduous. Results Here we present a genome-scale model of C. thermocellum metabolism, i SR432, for the purpose of establishing a computational tool to study the metabolic network of C. thermocellum and facilitate efforts to engineer C. thermocellum for biofuel production. The model consists of 577 reactions involving 525 intracellular metabolites, 432 genes, and a proteomic-based representation of a cellulosome. The process of constructing this metabolic model led to suggested annotation refinements for 27 genes and identification of areas of metabolism requiring further study. The accuracy of the i SR432 model was tested using experimental growth and by-product secretion data for growth on cellobiose and fructose. Analysis using this model captures the relationship between the reduction-oxidation state of the cell and ethanol secretion and allowed for prediction of gene deletions and environmental conditions that would increase ethanol production. Conclusions By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum and establish a strong foundation for rational strain design. In addition, we are able to draw some important conclusions regarding the underlying metabolic mechanisms for observed behaviors of C. thermocellum and highlight remaining gaps in the existing genome annotations.",
"conclusion": "Conclusions In this study, we applied constraint-based modeling to a genome-scale metabolic reconstruction of the cellulolytic, ethanologenic bacterium C. thermocellum in an effort to expedite research on this organism that has a high biofuel production potential. By incorporating genomic sequence data, network topology, and experimental measurements of enzyme activities and metabolite fluxes, we have generated a model that is reasonably accurate at predicting the cellular phenotype of C. thermocellum (at least for the environments shown in this study). The i SR432 model establishes a foundation for the integration and interpretation of large-scale systems biology data sets because the model's predictions can be further refined through the incorporation of thermodynamic constraints, gene regulatory data, and enzyme kinetics and because resulting models can be used for future strain design work involving combinations of gene modifications and chemical environments. Even at the level of studying single gene deletions in combination with chemical additions, analysis using the i SR432 model resulted in an interesting observation that ethanol production is influenced by the reduction-oxidation state of the cell. These observations illustrate the utility of this model as a predictive tool for rational manipulation of C. thermocellum metabolism.",
"discussion": "Discussion Current and future demands for renewable energy sources have spurred research in developing biofuels. One promising route for biofuel production is to use an organism-based bioprocess where cellulose could be converted to biofuel. One of the main challenges to this approach is that there are relatively few cellulolytic organisms capable of biofuel production, and none of these are especially well-characterized at present. Here we have implemented a computational modeling approach to study C. thermocellum , an anaerobic thermophile with high biofuel production potential. In this study, the development of a genome-scale metabolic model of C. thermocellum was used to provide a framework for analyzing the basic metabolic functions of C. thermocellum and improving its ethanol production capabilities. Overall, we report the construction of a genome-scale metabolic model of C. thermocellum , i SR432, and the accuracy of this model to predict cellular phenotypes (growth and fermentation product secretion) for growth on cellobiose and fructose in continuous and batch culture. Specific results of significance were: 1) the generation of a weighted amino acid representation of a cellulosome based upon proteomic data, 2) suggestions for additional genome annotations and identification of unanswered questions related to metabolism, and 3) identification of a general design principle related to intracellular reduction-oxidation balance that strongly influences the selection of gene deletions and chemical environments to increase ethanol production in C. thermocellum . We have constructed a genome-scale constraint-based model of C. thermocellum metabolism. The model accounts for 432 genes and includes 577 reactions involving 525 intracellular metabolites. Some thermodynamic constraints are placed on the metabolic solution space in the form of irreversible reactions. This reconstruction was tested by comparing computational predictions to experimentally measured growth rates and fermentation product secretion fluxes. By including as few as two experimentally determined values (substrate uptake rate and one fermentation-product secretion rate) as model constraints, we find that the model's predicted growth rate closely matches the experimentally observed value for continuous culture growth on cellobiose and fructose [ 32 ]. The addition of a third constraint (substrate uptake rate and two fermentation product secretion rates) gave reasonably accurate predictions for both continuous and batch culture growth. We should note that this step of testing the computational predictions to quantitative experimental data was a critical step to finalizing our current model. Prior to this step, our model calculated physiologically reasonable growth rates and by-product secretion profiles; however, there were several futile cycles that remained undetected until there were quantitative values of growth for comparison. The current model has no futile cycles resulting from computational artifacts (see Additional file 4 for details on removed reactions to prevent futile cycling). Comparison of model content from our model with that of models for C. acetobutylicum and S. cerevisiae reflects the expected relationships between the organisms; i.e., the two Clostridia models show much more overlap with each other than with the S. cerevisiae model. Shared functionality tends to be concentrated in central metabolism and nucleic acid metabolism, while primary differences tend to occur in how each organism can process starch and sucrose. Significantly, despite a large overlap between the clostridial models, significant functional differences are manifest between the models based on a relatively small number of reaction discrepancies. This points to the utility of constraint based models for making detailed functional predictions based on genome content, as well as the importance of correct genome annotation. In the process of constructing the model for C. thermocellum , we developed a weighted amino acid representation of a cellulosome that can be included as a component of the cellular objective required for growth. Experimental evidence suggests that the cellulosome, the extracellular structure responsible for cellulose degradation, comprises as much of 20% of the dry weight of C. thermocellum in certain conditions [ 40 ], and thus the metabolic demands associated with cellulosome production are significant. Cells experience a cost-benefit tradeoff when expressing proteins [ 41 ], thus we felt it necessary to formulate a cellulosome-specific component in our model. The current representation suggests that the increased production demand associated with the cellulosome decreases the growth rate by 4-17%. In vivo cellulosome production is of course dynamic, however, and the in silico representation is therefore designed to be tunable to available experimental data for different conditions by varying the relative contribution of the mass of the cellulosome to biomass or the ATP input required for cellulosome production. To our knowledge, our inclusion of an amino acid-weighted representation of a cell substructure, i.e. the cellulosome, as part of the biomass equation is the first such formulation used in constraint-based models. One of the useful features of genome-scale constraint-based models is that they can focus attention on areas of metabolism that are relatively unexplored, or illuminate high-priority areas for future research [ 42 ]. In light of i SR432, two examples of poorly characterized areas of metabolism in C. thermocellum that require further investigation were identified during the gap filling phase of model construction. These two areas are: the fate of succinate and the possibility of anaerobic respiration in this organism. Genomic evidence suggests an incomplete citric acid cycle in C. thermocellum . In our simulations, we found the citric acid cycle operates in the \"forward\" direction up through succinate, resulting in production and secretion of succinate. This behavior is not necessarily preferred by our model, unless constraints are placed on the production of other, more energetically favorable fermentation products. Secretion of succinate by C. thermocellum has been previously reported [ 31 ], although recent research does not show succinate as a major fermentation product of metabolism [ 32 , 37 ]. Thus, it is not clear if the citric acid cycle is complete or if it is incomplete resulting in production of succinate. In the current version of the model, all reactions of the citric acid cycle are present (based upon genome annotations and biochemical evidence) with the exception of succinate dehydrogenase. This question led to an experiment to assay for succinate dehydrogenase activity in C. thermocellum , however no activity was found. This leads us to believe that the citric acid cycle in C. thermocellum is not complete and there remains an open question regarding the cellular fate of intracellular succinate. If succinate is not secreted by C. thermocellum , or is secreted in very small amounts, this indicates that there must be intracellular fates of succinate that are not captured by current genome annotations or our current model. Possibilities include further processing via propanoate, glyoxalate, or tyrosine metabolism; though none of these seems likely on the basis of the available genomic evidence. Anaerobic respiration is another area of interest highlighted in our studies using i SR432. We found genomic evidence for NADH-quinone oxidoreductase. While there is no clear genomic evidence for a nitrate reductase, there is a C. thermocellum gene, Cthe_0199, that is a reciprocal best hit for a gene in the prokaryotic molybdopterin-containing oxidoreductase family [ 43 ], which includes nitrate reductases. Some, but not all, clostridia species are known to reduce nitrate [ 44 ]. There is at least one report specifically stating that C. thermocellum does not reduce nitrate under the conditions studied [ 31 ]. If C. thermocellum does not possess nitrate reductase or some other similar reaction, it is not clear how reduced quinone generated by NADH-quinone oxidoreductases might be reoxidized and it may be possible that the NADH-quinone oxidoreductase may be incorrectly annotated. One of our goals in constructing a model of C. thermocellum was to create a useful tool for strain design [ 10 , 22 , 45 ], so that interventions to increase the production of ethanol (or other desired fermentation products of interest) could be evaluated or designed in silico prior to experimental laboratory work. A simple illustration of this is provided by the results in Figure 3 , which shows non-lethal gene deletions expected to increase ethanol production. As expected, gene deletions that inhibit acetate production were found to increase the upper bound on ethanol secretion. We also found that eliminating reactions involved in the recycling of NADH back to NAD, namely NADH:ferredoxin oxidoreductase and ferredoxin hydrogenase, increase the upper bound on ethanol secretion. These results indicated a general relationship between the reduction-oxidation status of C. thermocellum and the production of ethanol. This finding was demonstrated more specifically in Figure 4 where the relationship between H 2 and ethanol production is shown. As H 2 production increases, ethanol production falls, eventually to zero. These results indicate that the capacity of C. thermocellum for ethanol production is strongly influenced by intracellular reduction-oxidation balance. Future strain design work in C. thermocellum likely needs to consider this aspect of C. thermocellum 's cellular physiology. This finding also points to the utility of using genome-scale metabolic models to facilitate the strain design process by reducing the workload associated with manually accounting for redox considerations. Additional computational analyses were conducted to study the effects of modifying the chemical environment of C. thermocellum . It was found that the addition of lactate or malate to the growth medium of C. thermocellum should induce a marked increase in ethanol secretion for a number of gene deletion strains. These results allude to the utility of specifying environmental conditions as a design parameter for engineering strains. In addition, the change in fermentation production secretion profile occurs naturally in C. thermocellum over the course of the its growth cycle as fermentation products secreted early in fermentation can influence what fermentation products are secreted in the late stages of fermentation."
} | 3,536 |
34366817 | PMC8339926 | pmc | 6,016 | {
"abstract": "Gated recurrent units (GRUs) are specialized memory elements for building recurrent neural networks. Despite their incredible success on various tasks, including extracting dynamics underlying neural data, little is understood about the specific dynamics representable in a GRU network. As a result, it is both difficult to know a priori how successful a GRU network will perform on a given task, and also their capacity to mimic the underlying behavior of their biological counterparts. Using a continuous time analysis, we gain intuition on the inner workings of GRU networks. We restrict our presentation to low dimensions, allowing for a comprehensive visualization. We found a surprisingly rich repertoire of dynamical features that includes stable limit cycles (nonlinear oscillations), multi-stable dynamics with various topologies, and homoclinic bifurcations. At the same time we were unable to train GRU networks to produce continuous attractors, which are hypothesized to exist in biological neural networks. We contextualize the usefulness of different kinds of observed dynamics and support our claims experimentally.",
"introduction": "1. Introduction Recurrent neural networks (RNNs) can capture and utilize sequential structure in natural and artificial languages, speech, video, and various other forms of time series. The recurrent information flow within an RNN implies that the data seen in the past has influence on the current state of the RNN, forming a mechanism for having memory through (nonlinear) temporal traces that encode both what and when . Past works have used RNNs to study neural population dynamics (Costa et al., 2017 ), and have demonstrated qualitatively similar dynamics between biological neural networks and artificial networks trained under analogs conditions (Mante et al., 2013 ; Sussillo et al., 2015 ; Cueva et al., 2020 ). In turn, this brings into question the efficacy of using such networks as a means to study brain function. With this in mind, training standard vanilla RNNs to capture long-range dependences within a sequence is challenging due to the vanishing gradient problem (Hochreiter, 1991 ; Bengio et al., 1994 ). Several special RNN architectures have been proposed to mitigate this issue, notably the long short-term memory (LSTM) units (Hochreiter and Schmidhuber, 1997 ) which explicitly guard against unwanted corruption of the information stored in the hidden state until necessary. Recently, a simplification of the LSTM called the gated recurrent unit (GRU) (Cho et al., 2014 ) has become popular in the computational neuroscience and machine learning communities thanks to its performance in speech (Prabhavalkar et al., 2017 ), music (Choi et al., 2017 ), video (Dwibedi et al., 2018 ), and extracting nonlinear dynamics underlying neural data (Pandarinath et al., 2018 ). However, certain mechanistic tasks, specifically unbounded counting, come easy to LSTM networks but not to GRU networks (Weiss et al., 2018 ). Despite these empirical findings, we lack systematic understanding of the internal time evolution of GRU's memory structure and its capability to represent nonlinear temporal dynamics. Such an understanding will make clear what specific tasks (natural and artificial) can or cannot be performed (Bengio et al., 1994 ), how computation is implemented (Beer, 2006 ; Sussillo and Barak, 2012 ), and help to predict qualitative behavior (Beer, 1995 ; Zhao and Park, 2016 ). In addition, a great deal of the literature discusses the local dynamics (equilibrium points) of RNNs (Bengio et al., 1994 ; Sussillo and Barak, 2012 ), but a complete theory requires an understanding of the global properties as well (Beer, 1995 ). Furthermore, a deterministic understanding of a GRU network's topological structure will provide fundamental insight as to a trained network's generalization ability, and therefore help in understanding how to seed RNNs for specific tasks (Doya, 1993 ; Sokół et al., 2019 ). In general, the hidden state dynamics of an RNN can be written as h t +1 = f ( h t , x t ) where x t is the current input in a sequence indexed by t , f is a nonlinear function, and h t represents the hidden memory state that carries all information responsible for future output. In the absence of input, h t evolves over time on its own: (1) h t + 1 = f ( h t ) where f (·) := f (·, 0 ) for notational simplicity. In other words, we can consider the temporal evolution of memory stored within an RNN as a trajectory of an autonomous dynamical system defined by Equation (1), and use dynamical systems theory to further investigate and classify the temporal features obtainable in an RNN. In this paper, we intend on providing a deep intuition of the inner workings of the GRU through a continuous time analysis. While RNNs are traditionally implemented in discrete time, we show in the next section that this form of the GRU can be interpreted as a numerical approximation of an underlying system of ordinary differential equations. Historically, discrete time systems are often more challenging to analyze when compared with their continuous time counterparts, primarily due to their more jumpy nature, allowing for more complex dynamics in low-dimensions (Pasemann, 1997 ; Laurent and von Brecht, 2017 ). Due to the relatively continuous nature of many abstract and physical systems, it may be of great use to analyze the underlying continuous time system of a trained RNN directly in some contexts, while interpreting the added dynamical complexity from the discretization as anomalies from numerical analysis (LeVeque and Leveque, 1992 ; Thomas, 1995 ; He et al., 2016 ; Heath, 2018 ). Furthermore, the recent development of Neural Ordinary Differential Equations have catalyzed the computational neuroscience and machine learning communities to turn much of their attention to continuous-time implementations of neural networks (Chen et al., 2018 ; Morrill et al., 2021 ). We discuss a vast array of observed local and global dynamical structures, and validate the theory by training GRUs to predict time series with prescribed dynamics. As to not compromise the presentation, we restrict our analysis to low dimensions for easy visualization (Beer, 1995 ; Zhao and Park, 2016 ). However, given a trained GRU of any finite dimension, the findings here still apply, and can be applied with further analysis on a case by case basis (more information on this in the discussion). Furthermore, to ensure our work is accessible we will assume a pedagogical approach in our delivery. We recommend Meiss (Meiss, 2007 ) for more background on the subject.",
"discussion": "6. Discussion Through example and experiment we indicated classes of dynamics which are crucial in expressing various known neural computations and obtainable with the 2D GRU network. We demonstrated the system's inability to learn continuous attractors, seemingly in any finite dimension, a structure hypothesized to exist in various neural representations. While the GRU network was not originally made as a neuroscientific model, there has been considerable work done showing high qualitative similarity between the underlying dynamics of neural recordings and artificial RNNs on the population level (Mante et al., 2013 ; Sussillo et al., 2015 ). Furthermore, recent research has modified such artificial models to simulate various neurobiological phenomenon (Heeger and Mackey, 2019 ). One recent study demonstrated that trained RNNs of different architectures and nonlinearities express very similar fixed point topologies to one another when successfully trained on the same tasks (Maheswaranathan et al., 2019a ), suggesting a possible connection in the dynamics of artificial networks and neural population dynamics. As such, an understanding of the obtainable dynamical features in a GRU network allow one to comment on the efficacy of using such an architecture as an analog of brain dynamics at the population level. Although this manuscript simplified the problem by considering the 2D GRU, a lot of research has resulted in interpreting cortical dynamics as low dimensional continuous time dynamical systems (Harvey et al., 2012 ; Mante et al., 2013 ; Cueva et al., 2020 ; MacDowell and Buschman, 2020 ; Zhao and Park, 2020 ; Flesch et al., 2021 ). This is not to say that most standard neuroscience inspired tasks can be solved with such a low dimensional network. However, demonstrating that common dynamical features in neuroscience can arise in low dimensions can aid in one's ability to comment on attributes of large networks. These attributes include features such as sparsity of synaptic connections. For example, spiking models exhibiting sparse connectivity have been shown to perform comparatively with fully connected RNNs (Bellec et al., 2018 ). Additionally, pruning (i.e., removing) substantial percentages of synaptic connections in a trained RNN is known to often result in little to no drop in the network's performance on the task it was trained on (Frankle and Carbin, 2019 ). This suggests two more examinable properties of large networks. The first is redundancy or multiple realizations of the dynamical mechanisms needed to enact a computation existing within the same network. For example, if only one limit cycle is sufficient to accurately perform a desired task, a trained network may exhibit multiple limit cycles, each qualitatively acting identically toward the overall computation. The second is the robustness of each topological structure to synaptic perturbation/pruning. For example, if we have some dynamical structure, say a limit cycle, how much can we move around in parameter space while still maintaining the existence of that structure? In a related light, the GRU architecture has been used within more complex machine learning setups to interpret the real-time dynamics of neural recordings (Pandarinath et al., 2018 ; Willett et al., 2021 ). These tools allow researchers to better understand and study the differences between neural responses, trial to trial. Knowledge of the inner workings and expressive power of GRU networks can only further our understanding of the limitations and optimization of such setups by the same line of reasoning previously stated, thereby helping to advance this class of technologies, aiding the field of neuroscience as a whole. The most compared RNN architecture to the GRU is LSTM, as GRU was designed as both a model and computational simplification of this preexisting design in discrete time implementation. LSTM, for a significant period of time, was arguably the most popular discrete time RNN architecture, outperforming other models of the time on many benchmark tasks. However, there is one caveat when comparing the continuous time implementations of LSTM and GRU. A one dimensional LSTM (i.e., a single LSTM unit) is a two dimensional dynamical system, as information is stored in both the system's hidden state and cell state (Hochreiter and Schmidhuber, 1997 ). With the choice of analysis we use to dissect the GRU in this paper, LSTM is a vastly different class of system. We would expect to see a different and more limited array of dynamics for an LSTM unit when compared with the 2D GRU. However, we wouldn't consider this a fair comparison. One attribute of the GRU architecture we chose to disregard in this manuscript was the influence of the update gate z ( t ). As stated in section 2, every element of this gate is bound to (0, 1) d . Since Equation (7) only has one term containing the update gate, [1 − z ( t )], which can be factored out, the fixed point topology does not depend on z ( t ), as this term is always strictly positive. The role this gate plays is to adjust the point-wise speed of flow, and therefore can bring rise to slow manifolds. Because each element of z ( t ) can become arbitrarily close to the value of one, regions of phase space associated with an element of the update-gate sufficiently close to one will experience seemingly no motion in the directions associated with those elements. For example, in the 2D GRU system, if the first element of z ( t ) is sufficiently close to one, the trajectory will maintain a near fixed value in x . These slow points are not actual fixed points. Therefore, in the autonomous system, trajectories traversing them will eventually overcome this stoppage given sufficient time. However, this may add one complicating factor for analyzing implemented continuous time GRUs in practice. The use of finite precision allows for the flow speed to dip below machine precision, essentially creating pseudo-attractors in these regions. The areas of phase space containing these points will qualitatively behave as attracting sets, but not by traditional dynamical systems terms, making them more difficult to analyze. If needed, we recommend looking at z ( t ) separately, because this term acts independently from the remaining terms in the continuous time system. Therefore, any slow points found can be superimposed with the traditional fixed points in phase space. In order to avoid the effects of finite precision all together, the system can be realized through a hardware implementation (Jordan and Park, 2020 ). However, proper care needs to be given in order to mitigate analog imperfections. Unlike the update gate, we demonstrated that the reset gate r ( t ) affects the network's fixed point topology, allowing for more complicated classes of dynamics, including homoclinic-like orbits. These effects are best described through the shape of the nullclines. We will keep things qualitative here as to help build intuition. In 2D, if every element of the reset gate weight matrix U r and bias b r is zero, nullclines can form two shapes. First is a sigmoid-like shape ( Figures 5A , 10 , 11 ; inferred limit cycle and line attractor), allowing them to intersect a line (or hyperplane in higher dimensions) orthogonal to their associated dimension a single time. The second is an s-like shape ( Figures 5B,C , 7 , 11 ; limit cycle), allowing them to intersect a line orthogonal to their associated dimension up to three times. The peak and trough of the s-like shape can be stretched infinitely as well ( Figure 2A ). In this case, two fo the three resultant seemingly disconnected nullclines associated with a given dimension can be placed arbitrarily close together ( Figure 3B ). Varying r ( t ) allows the geometry of the nullclines to take on several additional shapes. The first of these additional structures is a pitchfork-like shape ( Figures 3A,C , 9 ). By disconnecting two of the prongs from the pitchfork we get our second structure, simultaneously exhibiting a sigmoid-like shape and a U-like shape ( Figure 3C ). Bending the ends of the “U” at infinity down into ℝ 2 connects them, forming our third structure, an O-like shape ( Figure 3 periments; inferred ring attractor–orange nullcline). This O-like shape can then also intersect the additional segment of the nullcline, creating one continuous curve ( Figure 3 periments; inferred ring attractor–pink nullcline). One consequence of the reset-gate is the additional capacity to encode information in the form of stable fixed points. If we neglect r ( t ), we can obtain up to four sinks ( Figure 2A ), as we are limited to the intersections of the nullclines; two sets of three parallel lines. Incorporating r ( t ) increases the number of fixed points obtainable ( Figure 3A ). Refer to section 3 of the Supplementary Material to see how these nullcline structures lead to a vast array of different fixed point topologies. Several interesting extensions to this work immediately come to mind. For one, the extension to a 3D continuous time GRU network opens up the door for the possibility of more complex dynamical features. Three spatial dimensions are the minimum required to experience chaotic dynamics in nonlinear systems (Meiss, 2007 ), and due to the vast size of the GRU parameter space, even in low dimensions, such behavior is probable. Similarly, additional types of bifurcations may be present, including bifurcations of limit cycles, allowing for more complex oscillatory behavior (Kuznetsov, 1998 ). Furthermore, higher dimensional GRUs may bring rise to complex center manifolds, requiring center manifold reduction to better analyze and interpret the phase space dynamics (Carr, 1981 ). While we considered the underlying GRU topology separate from training, considering how the attractor structure influences learning can bring insight into successfully implementing RNN models (Sokół et al., 2019 ). As of yet, this topic of research is mostly uncharted. We believe such findings, along with the work presented in this manuscript, will unlock new avenues of research on the trainability of recurrent neural networks and help to further understand their mathematical parallels with biological neural networks."
} | 4,253 |
26579074 | PMC4620420 | pmc | 6,018 | {
"abstract": "Yellowstone Lake (Yellowstone National Park, WY, USA) is a large high-altitude (2200 m), fresh-water lake, which straddles an extensive caldera and is the center of significant geothermal activity. The primary goal of this interdisciplinary study was to evaluate the microbial populations inhabiting thermal vent communities in Yellowstone Lake using 16S rRNA gene and random metagenome sequencing, and to determine how geochemical attributes of vent waters influence the distribution of specific microorganisms and their metabolic potential. Thermal vent waters and associated microbial biomass were sampled during two field seasons (2007–2008) using a remotely operated vehicle (ROV). Sublacustrine thermal vent waters (circa 50–90°C) contained elevated concentrations of numerous constituents associated with geothermal activity including dissolved hydrogen, sulfide, methane and carbon dioxide. Microorganisms associated with sulfur-rich filamentous “streamer” communities of Inflated Plain and West Thumb (pH range 5–6) were dominated by bacteria from the Aquificales, but also contained thermophilic archaea from the Crenarchaeota and Euryarchaeota. Novel groups of methanogens and members of the Korarchaeota were observed in vents from West Thumb and Elliot's Crater (pH 5–6). Conversely, metagenome sequence from Mary Bay vent sediments did not yield large assemblies, and contained diverse thermophilic and nonthermophilic bacterial relatives. Analysis of functional genes associated with the major vent populations indicated a direct linkage to high concentrations of carbon dioxide, reduced sulfur (sulfide and/or elemental S), hydrogen and methane in the deep thermal ecosystems. Our observations show that sublacustrine thermal vents in Yellowstone Lake support novel thermophilic communities, which contain microorganisms with functional attributes not found to date in terrestrial geothermal systems of YNP.",
"introduction": "Introduction Submarine and sublacustrine thermal vents are found throughout the world and support an enormous diversity of life. Hydrothermal vent fluids often contain high concentrations of reduced constituents such as iron, sulfide, hydrogen, methane, arsenic, and/or ammonia that provide numerous possibilities for chemolithotrophic metabolism (Reysenbach et al., 2000 ; Amend and Shock, 2001 ; Coumou et al., 2008 ), as well as carbon dioxide important for supporting autotrophic organisms (Lovalvo et al., 2010 ). Hydrothermal discharge creates complex and dynamic temperature and geochemical gradients upon mixing with colder waters; the microorganisms that colonize different niches surrounding hydrothermal vents are of considerable interest in marine biology (e.g., Van Dover et al., 2001 , 2007 ; Harmer et al., 2008 ), in part due to the potential microbial linkages with element cycling as well as the evolutionary implications of thermophilic organisms in marine settings (Reysenbach et al., 2000 ). The presence of eukaryotic mutualists adjacent to hydrothermal vents is often made possible by microbial symbionts capable of chemolithotrophic metabolism using reduced constituents present in vent fluids (Harmer et al., 2008 ; Setoguchi et al., 2014 ). Consequently, thermal vent microorganisms often conduct redox transformations and/or provide a source of nutrients important in the evolution of eukaryotes. Prior mapping and detailed geophysical analysis of Yellowstone Lake has provided critical information on the volcanology, geologic history and current location of major thermal activity on the lake floor (Morgan et al., 2003 ; Morgan and Shanks, 2005 ; Shanks et al., 2005 ). Prior sampling of hydrothermal vents in Yellowstone Lake provided important background information regarding the location and characteristics of different vent types (Johnson et al., 2003 ; Morgan et al., 2003 , 2007 ; Morgan and Shanks, 2005 ; Shanks et al., 2005 ). The northern region of Yellowstone Lake is one of the most seismically active areas in Yellowstone Park and supports high geothermal heat fluxes of 500–2000 mW m −2 (Figure 1 ). Mary Bay itself was created as a result of an explosion crater that occurred approximately 0.2 Ma (Wold et al., 1977 ), and numerous other smaller features in this region attest to a dynamic and recent volcanic history (Morgan et al., 2009 ). The isotopic and geochemical composition of Yellowstone lake waters, vent waters and tributaries have shown that elevated levels of numerous trace elements (As, Se, B, Li, Cs, Ga) in Yellowstone Lake are due to hydrothermal inputs that represent ~10% of the total chloride flux from all of the geothermal features in YNP (Shanks et al., 2005 , 2007 ; Balistrieri et al., 2007 ). Moreover, Cl − vs. 2 H 2 O plots place submerged vents in Yellowstone Lake on a mixing line between lake bottom-water and thermal fluids, which have an approximate temperature of 220°C (Shanks et al., 2005 ). High levels of trace elements, major nutrients, and/or energy sources near vent discharge have been shown to influence the diversity and productivity of biological communities in Yellowstone Lake (Lovalvo et al., 2010 ; Clingenpeel et al., 2011 , 2013 ; Kan et al., 2011 ; Yang et al., 2011 ). Figure 1 Bathymetric map (Morgan and Shanks, 2005 ) of Yellowstone Lake showing heat flux iso-lines (mW/m 2 ) (Morgan et al., 1977 ) and sampling locations of thermal vents ( Table 1 ) discussed in the current study (IP, Inflated Plain; WT-DV, West Thumb Deep Vents; WT-OV, West Thumb Otter Vent; EC, Elliott's Crater; MB, Mary Bay; SA, Southeast Arm; see Table S1 for GPS coordinates) . Efforts to characterize microbial communities from several vent sites in Yellowstone Lake using modest bacterial 16S rRNA gene surveys have shown that thermophilic bacteria from the order Aquificales were important in sulfidic habitats (Yang et al., 2011 ). Sulfur oxidizing Proteobacteria were also important in several vent sites, including organisms related to Thiovirga spp., Thiobacillus spp., and Sulfuricurvum spp. Geochemical analyses of the higher-temperature (i.e., >50°C), deeper (>49 m) vent sites (3) confirmed high levels of sulfide and other reduced sulfur species, which upon mixing with oxygenated lake water, provide habitats suitable for sulfur-oxidizing microbial communities, and which support significant rates of dark CO 2 fixation (Yang et al., 2011 ). The prior geochemical work on Yellowstone Lake thermal vents (Shanks et al., 2005 ; Balistrieri et al., 2007 ), as well as efforts to characterize microorganisms present in these communities (Yang et al., 2011 ), or in filtered vent fluids (Clingenpeel et al., 2011 , 2013 ; Kan et al., 2011 ), suggested that thermal vents in Yellowstone Lake contain thermophilic communities whose functional attributes can be correlated with pronounced chemosynthetic gradients. Moreover, several sublacustrine vents in Yellowstone Lake exhibit unique chemical signatures that support novel assemblages of both Bacteria and Archaea . Here we report an integrated study of hydrothermal vent geochemistry, and associated molecular and microscopic analysis of microbial communities from several of the major vent types in Yellowstone Lake (YNP, USA). The primary objectives of the study were to (i) determine the geochemical composition of hydrothermal vent fluids and predominant solid phases associated with hydrothermal vents in Yellowstone Lake, (ii) identify predominant thermophilic microbial populations inhabiting major vent types in Yellowstone Lake using both 16S rRNA gene and random shotgun sequencing, and (iii) compare differences in functional genes observed in metagenome sequence obtained from vents exhibiting different geochemical signatures. Geochemical analysis indicated that thermal vents in Yellowstone Lake contain high concentrations of dissolved gases including H 2 S, H 2 , CH 4 , and CO 2 , as well as various trace elements and hydrogen ions (pH values ranged from 5 to 6.4 in deep vents, compared to bulk lake water pH = 7.0). Our results showed a definitive linkage between vent chemistry, microbial community structure, and associated metabolic attributes of microorganisms supported by high-temperature systems in Yellowstone Lake.",
"discussion": "Results and discussion Geochemical analysis of sublacustrine thermal vents in YNP Aqueous samples Temperature values measured at the sampling end of the suction arm (Table 1 ) confirmed that all vent waters collected with the ROV (Figure S1 ) had received significant inputs of hydrothermal water, and/or had been heated due to adjacent thermal activity. The large range in vent temperature(s) at a single sampling location was due to the dynamics of mixing with surrounding lake water at temperatures of 8–10°C. In most cases, stable temperatures above 60°C were maintained for extended measurement periods of 1–2 h during fluid collection. The concentrations of many constituents considered signatures of geothermal activity, such as dissolved CO 2 , H 2 , H 2 S, and CH 4 were considerably higher in thermal vent waters relative to background lake water (e.g., Southeast Arm, Table 1 ). The deep thermal vents were all mildly acidic compared to bulk lake water, ranging from pH 5.1 at Mary Bay (MB), 5.2–5.6 at Inflated Plain (IP), 5.9–6.2 at West Thumb (WT), and 6.2–6.4 at Elliot's Crater (EC). A shallow (4.3 m) “alkaline siliceous” thermal vent on the west side of WT (i.e., the Otter Vent) exhibited a pH ~ 8.2. Lower pH values at MB and IP were correlated with higher concentrations of Fe and Al (Table S1 ), consistent with mineral solubility as a function of pH. Other key indicator constituents of geothermal inputs were observed at concentrations significantly higher than background lake water (>5–10x), and included F, NH 4 , As, Sb, W, Mo, Li, Cs, B, and/or Na (Table S1 ). Concentrations of major cations (Ca, Mg, K) and anions (Cl, SO 4 ) were generally similar in vent vs. lake waters, although vent waters at WT revealed high levels of Cl and SO 4 , as well as Na. Table 1 Key geochemical characteristics a , temperature values and sample depths of sublacustrine thermal vent waters (and lake water from the Southeast Arm) obtained from Yellowstone Lake using the remotely operated vehicle (ROV) during September 2007 and 2008 . Sample Depth Temp pH DIC a CO 2 (aq) b DS a O 2 (aq) b CH 4 (aq) b H 2 (aq) b Sample Location m °C mM μM nM ID c Date Inflated Plain 30 92–94 5.6 8.5 8.1 633 bd 21.8 414 329-Sy-1 9/9/2007 32 70–76 5.6 4.1 3.2 463 25 21.2 4837 330-Sy-1 9/10/2007 32 40–45 5.2 3.1 3.1 230 bd 20.9 1031 348-Sy-P 9/11/2008 30 40–60 5.2 3.2 3.2 266 bd 22.5 1430 348-Sy-S 9/11/2008 33.6 44–52 5.5 1.2 1.2 85 25 6.7 1023 359-VC 9/16/2008 33.6 41–49 5.7 1.1 1.1 111 bd 5.4 1974 359-Sy-S 9/16/2008 West Thumb 52 60–66 6.2 4.7 1.6 2 113 6.4 41 339-VC 9/18/2007 Deep Vent 52 60–66 6.2 nd d 1.8 bd e bd 7.2 63 339-Sy 9/18/2007 52 60-76 6.2 1.3 0.4 1 bd 14.5 30 341-Sy-1 9/19/2007 54 66 6.1 2.5 1.0 8 188 4.6 102 343-Sy-1 9/19/2007 53.2 40-53 6.1 nd 2.1 10 147 5.6 23 369-VC 9/20/2008 53.2 38 5.9 nd 2.0 10 238 6.8 33 369-Sy-P 9/20/2008 53.2 62-66 5.9 nd 3.0 13 210 10.5 41 369-Sy-S 9/20/2008 Mary Bay 49.6 30-57 5.1 4.0 3.3 172 bd 13.7 454 335-Sy-S 9/16/2007 52.3 62-66 5.1 6.4 4.5 385 bd 17.9 511 336-Sy-1 9/16/2007 52.3 73-74 5.1 5.9 6.0 433 bd 20.4 100 336-Sy-2 9/16/2007 50.5 65-70 5.0 3.8 3.8 bd bd 28.1 2984 349-Sy-P 9/12/2008 50.5 62-70 5.4 1.8 1.8 123 94 12.4 2797 349-Sy-S 9/12/2008 Elliot's Crater 14.1 71 6.4 1.4 0.5 43 202 4.4 688 351-Sy-S 9/13/2008 14.1 71 6.2 1.6 0.7 54 190 1.9 672 351-Sy-P 9/13/2008 14.1 63-68 6.4 1.3 0.5 40 120 2.3 660 352-VC 9/14/2008 West Thumb 4.3 65-70 8.4 0.8 0.0 bd 47 2.1 551 332-Sy 9/11/2007 Otter Vent 4.3 63-68 8.4 0.7 0.0 bd 26 0.1 43 333-VC 9/12/2007 Southeast Arm 2.5 10.6 7.0 0.6 0.013 bd 234 0.1 10 344 9/20/2007 3 11 7.0 0.6 0.014 bd 313 0.2 47 354 9/15/2008 17 10.5 7.1 0.6 0.019 bd 344 0.1 23 356 9/15/2008 a DIC, dissolved inorganic C; DS, dissolved sulfide; other constituents given in Table S1 . b Dissolved gas species determined using headspace GC, aq, aqueous. c Sy, ROV syringe, P, port side, S, starboard side, VC, vent carboy/peristaltic pump. d nd, not determined. e bd, below detection; detection limit DS = 0.3 μM; O 2 = 3 μM. Dissolved gas [H 2 S(aq), CO 2 (aq), H 2 (aq), and CH 4 (aq)] concentrations from thermal vents were one to two orders of magnitude higher than in background lake water (Table 1 ), and were considerably higher than measured in terrestrial sites of YNP (Spear et al., 2005 ; Inskeep et al., 2013a ). Vent waters from MB and IP contained the highest levels of total dissolved sulfide (DS), H 2 (aq), and CH 4 (aq), and were also the most acidic waters found in the study. Although, the concentrations of dissolved gases varied across different sample types collected for a given vent, the measurements were reasonably stable considering the sampling challenges presented under these circumstances (i.e., rapid mixing with bulk lake water). The large flux of H 2 S(g) from the IP vent region resulted in concentrations of DS well-above detection (e.g., 3–5 μM) in several surface (0–10 cm) lake samples obtained within discharge zones at IP. Microscopy and solid phase analysis Scanning electron microscopy (FE-SEM) of vent biomass provided considerable insight regarding the characteristics of each sample, and the potential processes responsible for the formation of filamentous structures. Images of the sulfur-rich streamers from IP (Figure S2 ) reveal coccoid, rod-shaped, and filamentous organisms contained in a complex extracellular matrix including rhombohedral crystals of elemental S (Figure 2A ). Extracellular substances were a dominant feature observed in streamers from IP, and although the exact composition of these materials is not known, the resultant “streamer structures” are very resistant to dispersion and/or disaggregation. West Thumb streamers were notably more complex, and contained diverse cellular structures, less elemental S, and more diatom shells. The vent sediments collected from MB and EC also contained numerous diatom shells intermixed with a complex suite of siliceous minerals, aluminosilicates and organic material (Figure 2B ). The extracellular matrix evident in the thermophilic IP streamers envelopes bundles of individual filaments and sulfur crystals into dense “liquid-like” structures that exhibit significant cohesion (Figure S2 ). Figure 2 (A) Scanning electron micrographs of thermal streamer communities obtained from 30 to 33 m vents in the Inflated Plain region, Yellowstone Lake (Sample ID). All scale bars = 1 μm. (B) Scanning electron micrographs of thermal vent biomass samples obtained from vent sites at West Thumb deep (Sample ID 339, 342; 2007), Elliot's Crater (351; 2008), and Mary Bay (349; 2008). Sediments associated with thermal vents show accumulation of diatom shells (e.g., Mary Bay, 349, lower right), which were also trapped in filamentous streamer communities (e.g., West Thumb, 369, lower left). Microbial community structure and function Long-fragment (>1000 bp) archaeal and bacterial 16S rRNA gene sequences indicated the major types of thermophilic microorganisms present in vent biomass (Table 2 , Figure 3 ). Sulfurihydrogenibium spp. (order Aquificales) were a significant fraction of the bacterial populations observed in sulfur streamers from IP and WT, and these organisms are also found in sulfidic geothermal springs of YNP (Nakagawa et al., 2005 ; Reysenbach et al., 2005 ; Inskeep et al., 2010 ; Takacs-Vesbach et al., 2013 ). Other bacteria observed in streamer communities from IP and WT included Caldisericum (Candidate Division OP5), Geothermobacterium, Sulfuricurvum, Thiovirga , and Thiobacillus spp. (Proteobacteria), all of which are often found in sulfidic environments (Inskeep et al., 2005 ; Ito et al., 2005 ; Mori et al., 2009 ; Han et al., 2012 ). Deep (~50 m) vents at WT were the only samples to exhibit relatives of Methylothermus thermalis (Methylococcales), and these sequences comprised ~10, 28, and 64% of the bacteria observed in 3 independent vents from this region (Table 2 ). Table 2 Summary of predominant long-fragment bacterial 16S rRNA gene sequences (nt > 1200 bp) obtained from thermal vent biomass samples from Yellowstone Lake using clone-library analysis ( n ~ 48 per site) . Location year (ID) Closest cultivated relative a Taxonomic group b Percent library Clone ID IDY (%) Relative NCBI No . Inflated Plain Sulfurihydrogenibiumsp. Y04ACS1 Aquificales 83 329-10-20 ~99 AM259493.1 Geothermobacterium ferrireducens Thermodesulfobacteria 5 329-5 98.6 AF411013.1 2007 (329S) Caldisericum exile AZM16c01 Candid. Div. OP5 5 329-21 98.3 NR_075015.1 Thiovirga sulfuroxydans Chromatiales 5 329-22 97.3 NR_040986.1 2008 (348S) Sulfurihydrogenibium sp. Y04ACS1 Aquificales 100 348-5 >99 AM259493.1 2008 (359S) Sulfurihydrogenibium sp. Y04ACS1 Aquificales 100 359-5 >99 AM259493.1 West Thumb Sulfurihydrogenibium yellowstonense Aquificales 30 339-15,16 >99 JQ346738.1 Thiovirga sulfuroxydans Chromatiales 20 339-20, 24 97.3 NR_040986.1 2007 (339) Methylothermus thermalis MYH Methylococcales 10 339-13 92.8 NR_043209.1 Caldisericum exile AZM16c01 Candid. Div. OP5 10 339-17 98.5 NR_075015.1 2007 (342) Methylothermus thermalis MYH Methylococcales 64 342-4 93.0 NR_043209.1 Sulfurihydrogenibium sp. Y03AOP1 Aquificales 10 342-12 99.4 NR_074557.1 Thiobacillus denitrificans Hydrogenophilales 10 342-5 91.0 NR_074417.1 Curvibacter sp. PL21 Burkholderiales 3 342-7 98.9 KF206393.1 Thermus aquaticus Thermales 3 342-9 95.4 NR_025900.1 Bellilinea sp. clone 96 Nov. Chloroflexi 3 342-8 < 95 JQ183076.1 2008 (369S) Methylothermus thermalis MYH Methylococcales 28 369-20 92.8 NR_043209.1 Sulfurihydrogenibium sp. Y04ACS1 Aquificales 25 369-21 99.6 AM259493.1 Thiobacillus dentrificans Hydrogenophilales 6 369-25 96.1 NR_074417.1 Geothermobacterium ferrireducens Thermodesulfobacteria 3 369-19 98.3 AF411013.1 Fervidobacterium sp. CBS-3 Thermotogales 3 369-18 91.3 EF222230.1 Syntrophomonas palmitatica MPA Syntrophobacterales 3 369-22 94.5 NR_041528.1 Thermodesulforhabdus norvegica Syntrophobacterales 3 369-23 91.8 NR_025970.1 Rhodoferax ferrireducens Burkholderiales 3 369-27 97.5 NR_074760.1 Thermus aquaticus Thermales 3 369-24 98.0 NR_025900.1 Desulfomicrobium thermophilum P6.2 Uncl. Proteobacteria 3 369-16 96.8 NR_042924.1 Pelosinus sp. UFO1 Firmicutes 3 369-268 94.0 DQ295866.1 Mary Bay Caldisericum exile AZM16c01 Candid. Div. OP5 12 349-20 89.9 NR_075015.1 Thermanaerothrix daxensis GNS-1 Chloroflexi 12 349-10 92.9 HM596746.1 2008 (349S) Cystobacter violaceus Cbvi34 Myxococcales 12 349-11 93.2 KF267724.1 Desulfosarcina cetonica Desulfurobacteriales 12 349-12 92.1 NR_028896.1 Ornatilinea apprima Chloroflexi 8 349-15 88.9 NR_109544.1 Prosthecobacter fluviatilis Verrucomicrobiales 8 349-21 83.5 NR_041608.1 Methylobacter psychrophilus Z-0021 Methylococcales 8 349-17 98.5 NR_025016.1 Geobacter daltonii Desulfomonadales 4 349-13 84.4 NR_074916.1 Syntrophus aciditrophicus SB Syntrophobacterales 4 349-19 83.2 NR_102776.1 Aquihabitans daechungensis Acidimicrobiales 4 349-14 93.5 NR_132289.1 Elliot's Crater Thiobacillus thioparus DSM505 Hydrogenophilales 57 351-8 96.8 NR_117864.1 Bellilinea caldifistulae Chloroflexi 14 351-1 90.4 NR_041354.1 2008 (351) Sulfurihydrogenibium sp. Y04ACS1 Aquificales 9 351-5 98.9 AM259493.1 Pelobacter massiliensis DSM6233TN Desulfuromonadales 4 351-4 85.3 FR749901.1 Denitratisoma sp. TSA61 Rhodocyclales 4 351-132 94.9 AB542411.1 Thermomonas hydrothermalis SGM-6 Xanthomonadales 2 351-7 98.0 NR 025265.1 Caldisericum exile AZM16c01 Candid. Division OP5 2 351-130 97.4 NR 075015.1 Otter Vent Fervidobacterium changbaicum Thermotogales 27 332-7 94.7 NR 043248.1 Synechococcus sp. TS-97 B' Cyanobacteria 11 332-16 99.7 AY884056.1 2007 (332) Anaeromyxobacter sp. K Myxococcales 11 332-13 86.0 NR 074969.1 Thermodesulfoba. hydrogeniphilum Thermodesulfobacteria 11 332-17 79.6 NR 025146.1 Chloroflexus sp. strain 396-1 Chloroflexi 8 332-5 98.1 AJ308498.1 Thiobacter subterraneus C55 Burkholderiales 8 332-18 92.1 NR 024834.1 Acidobacteria bacterium KBS 96 Acidobacteriales 5 332-3 91.8 FJ870384.1 Fischerella sp. JSC-11 Cyanobacteria 5 332-12 99.9 HM636645.1 Anaerolinea thermophila UNI-1 Anaerolineales 3 332-4 98.6 AP012029.1 a In some cases, clones are listed due to distant cultivated relatives. All Yellowstone Lake 16S rRNA gene sequences are deposited in GenBank [Accession Numbers KT453543 - KT453636 ]. b Major phylum, order, or family. Figure 3 Phylogenetic tree (16S rRNA gene sequences) of the domain Archaea including long-fragment sequences observed in thermal vent microbial communities from Yellowstone Lake (neighbor-joining tree; bootstrap values reported based on 1000reps Log Det.) . All long-fragment 16S rRNA gene sequences from Yellowstone Lake are deposited in GenBank ( KT453543 - KT453636 ). The sulfur streamers from WT contained 16S rRNA gene sequences representing 6 major lineages in the Archaea (Figure 3 ), including members of the Korarchaeota and Euryarchaeota, which were notably absent in replicate (temporal and spatial) streamer samples from IP. Archaea present in the sulfur streamers from IP were dominated by members of the Crenarchaeota (including the Desulfurococcales and Thermoproteales), as well as a novel group of Euryarchaeota (related to the Thermoplasmatales), which are also observed in sulfur sediments of terrestrial YNP springs (Inskeep et al., 2013b ). The MB sediments also contained undescribed archaeal populations including members of the Aigarchaeota, Thaumarchaeota, and Euryarchaeota (primarily relatives of methanogens), although no Crenarchaeota were observed. Compared to IP streamers, larger contributions of non-thermophilic bacteria were detected in samples from WT, MB, and EC. Bacterial sequences from MB sediments revealed an extensive diversity of different Proteobacteria, many of which are more closely related to moderate thermophiles and/or mesophiles often found in extreme sulfur and/or iron-rich habitats (e.g., Ito et al., 2005 ). The greater number of different bacterial sequence types observed in MB and EC sediments (Table 2 ) was consistent with sampling constraints at these locations, which resulted in collection of a significant amount of sediment adjacent to the vent exit walls. Bacterial sequences from the shallow phototrophic communities (pH 8.2) at the WT-Otter Vent (OV) corresponded to two major cyanobacterial groups ( Synechococccus and Fisherella spp.), different members of the Chloroflexi, as well as major contributions (~27% of the clone library) from a novel Thermotogales population ( Fervidobacterium spp.) (Table 2 ). Pyro-tag sequencing Four vent biomass samples were subjected to more intensive 16S rRNA gene sequencing as well as random shotgun sequencing. The majority of phylotypes observed using pyro-tag sequencing (Table 3 ) of IP streamers (2 sites), WT streamers, and MB sediments were also found using long-fragment sequence analysis, and provided corroborative evidence of the major taxonomic groups present. Aquificales-like sequences (i.e., Sulfurihydrogenibium sp.) dominated the bacterial 16S rRNA gene libraries (74–84%) obtained from two IP sulfur streamers (Figure 4 ). Conversely, the WT streamers exhibited significantly greater bacterial diversity and contained only 10% Aquificales (Table 3 ), which is consistent with lower DS and H 2 (aq) relative to the vents at IP (Table 1 ). Mary Bay vent sediments contained very few Aquificales sequences, consistent with the lack of any notable streamers at this site, and the significant contribution from mesophilic organisms. Populations related to Caldisericum exile (Mori et al., 2009 ; candidate phylum OP5) were observed in all samples, but especially in association with the sulfur streamers at IP (Figure 4 ). Table 3 Major taxonomic groups (fraction of total bacterial or archaeal sequences) in vent-associated microbial communities determined using pyro-tag 16S rRNA gene sequencing of amplicons generated with universal bacterial (top) and archaeal (bottom) primer sets (Clingenpeel et al., 2011 , 2013 ; Kan et al., 2011 ) . Taxonomic groups a Bacteria Site name IP 348S IP 359S WT 369S MB 349S Aquificae 83.8 73.9 10.3 0.4 Caldiserica 5.8 4.9 0.7 2.6 Bacteroidetes 1.0 2.8 12.7 23.9 Proteobacteria 4.5 13.1 25.3 13.6 Acidobacteria 2.2 0.5 1.4 3.6 Chloroflexi 0.1 0.0 6.4 7.4 Thermotogae 0.0 0.1 7.1 0.1 Actinobacteria 0.3 0.6 1.3 5.0 Deinococcus-Thermus 0.0 0.0 4.5 0.0 Firmicutes 0.1 0.3 3.3 2.1 Cyanobacteria 0.1 0.0 1.0 12.7 Thermodesulfobacteria 0.8 0.1 2.4 0.1 Chlorobi 0.0 0.0 2.2 1.1 Planctomycetes 0.0 0.1 1.3 1.1 Gemmatimonadetes 0.0 0.0 0.5 1.6 Nitrospira 0.0 0.0 1.0 0.3 Unclassified Bacteria 1.0 2.7 15.6 21.5 Total b 99.7 99.1 97 97.1 n 27540 32471 27338 25753 Archaea IP IP WT MB CRENARCHAEOTA Desulfurococcales 48.8 12.5 17.4 17.0 Thermoproteales 49.2 86.2 21.9 14.2 Sulfolobales 0.2 0.2 0.0 0.1 Other “Crenarchaeota” 1.8 0.9 2.4 22.8 EURYARCHAEOTA Methanomicrobiales 0.0 0.0 0.3 0.6 Thermoplasmatales 0.0 0.0 0.5 0.9 Novel Euryarchaeota 0.0 0.1 41.6 12.2 Korarchaeota 0.0 0.0 9.7 23.1 Unclassified Archaea 0.0 0.0 5.4 8.7 Total b 100.0 99.9 99.2 99.6 n 24191 13945 13495 26382 a RDP training set 9, RDP Naive Bayesian rRNA Classifier version 2.5, May 2012 Classifications performed March 18, 2013. Novel Crenarchaeota include what was referred to as “Marine Crenarchaeota,” now established within the Candidate phylum Thaumarchaeota. b Total = percent of total sequences (n). Figure 4 Phylogenetic classification of short fragment bacterial 16S rRNA gene sequences from two different sulfur streamer communities from Inflated Plain (depth ~ 30–33 m; pH ~ 5.6) obtained using pyro-tag sequencing (sequences classified using RDP Naïve Bayesian rRNA Classifier version 2.5; also see Table 3 ) . Other major groups of Bacteria varied with vent sites, but included members of the Bacteroidetes, Proteobacteria (the Epsilon group was more important in IP whereas Beta and Delta groups were more important in WT and MB), Thermotogae and Deinococcus-Thermus (7.2 and 4.5% in WT streamers), Acidobacteria (4% in MB sediments), Actinobacteria (5.4% in MB sediments), Thermodesulfobacteria (2.4% in WT), Planctomycetes ~2% in WT and MB), as well as members of the Chloroflexi (~6–8% in WT and MB sediments) (Table 3 ). It is unlikely that Chloroflexi-like sequences are contributed from organisms conducting photosynthesis at these depths; phylogenetic placement of long-fragment 16S rRNA sequences that were highly related to the shorter pyro-tag reads suggest that many of the Chloroflexi sequences were contributed by relatives of anaerobic, heterotrophic strains (Yamada et al., 2007 ; Klatt et al., 2013 ) (Table 3 ). Different types of Archaea were observed across sites (Table 3 ), and the major groups identified using pyro-tag sequencing were also observed in long-fragment clone libraries (e.g., Table 2 , Figure 3 ). The highly sulfidic and H 2 -rich IP streamers (pH ~ 5.2–5.6) exhibited a consistent signature of Crenarchaeota (>99% of archaeal reads), including members of the Thermoproteales ( Pyrobaculum and Thermofilum -like populations) and Desulfurococcales ( Desulfurococcus and Acidilobus -like sequences; Jay et al., 2014 ). Very few Sulfolobales sequences were observed, which is expected given the pH range of these vent communities (pH 5–6) (Macur et al., 2013 ; Jay et al., 2014 ). Members of the Korarchaeota were found primarily in the less sulfidic and higher pH streamers from WT, as well as in sediments from MB (Table 3 ). A significant number of novel euryarchaeotal sequences were observed in WT and MB, and represent several novel methanogens, an undescribed group related to the order Thermoplasmatales (~85% nt identity, Figure 3 ), as well as members of the Thaumarchaeota and Aigarchaeota (Brochier-Armanet et al., 2008 ; Nunoura et al., 2011 ). Long-fragment clone libraries also indicated the presence of different types of Euryarchaeota and Thaumarchaeota in WT streamers and MB sediments (Figure 3 ), including relatives of both low-temperature thaumarchaea (Hatzenpichler, 2012 ) as well as thermophilic clades (Beam et al., 2014 ). The korarchaeotal sequences observed using pyro-tag analysis (~10–23% of WT and MB pyro-tag sequences) corresponded to long-fragment 16S rRNA gene sequences, which were observed at several WT vent sites in both 2007 and 2008 (Figure 3 ). Metagenome sequence analysis Random shotgun sequence (average read length ~400 bp) obtained from four vent sites (IP, WT, and MB) was analyzed using Blastx (NCBI) and G + C content (%) to examine the predominant populations present in each site (Figure 5 ). The random sequence data indicated a lower abundance of archaea relative to bacteria in all vents sampled, representing from less than 5% of the total sequences in three of the four vent sites up to nearly 30% in one of the sulfur streamers from IP (348S). The major phylotypes identified with random sequence were also consistent with those observed using amplification techniques. For example, random sequence reads from two different streamer communities from IP were dominated by sequences related to Sulfurihydrogenibium, Caldisericum , and other Proteobacteria (Figure 5 ). The streamer communities from WT were dominated by sequences related to members of the Bacteroidetes, Aquificales, and Proteobacteria, and the sediments from MB contained a diverse assemblage of distant relatives of the Bacteroidetes (lower G + C), Proteobacteria (higher G + C), Chlamydiae/Verucomicrobia, and Actinobacteria. Much of the random shotgun sequence from MB (and to a lesser extent in WT) was not sufficiently similar to reference organisms (NCBI) to assign individual sequence reads to specific genera. Figure 5 Random shotgun sequence reads from four Yellowstone Lake thermal vent microbial communities plotted as a function of G + C content (%) and subjected to phylogenetic analysis using blast (90% identity) . A significant number of sequence reads were not related to bacteria or archaea in current public databases. The amount of assembled genome sequence (Table S2 ) obtained from the four vent sites was inversely correlated with the number of dominant sequence types observed using 16S rRNA gene inventories. For example, the higher percent of reads assembled from IP streamers (348S and 359S) resulted in larger contigs with higher sequence coverage (Table S2 ). Phylogenetic assignment of 16S rRNA genes obtained from assembled sequence (Table S3 ) was consistent with populations observed using 16S rRNA gene-only approaches (Tables 2 , 3 , Figures 3 , 4 ). Consequently, the sequence assemblies from IP and WT represent an excellent opportunity for linking specific metabolic genes with known phylotypes. Functional gene analysis The predominant energy cycling reactions mediated by microorganisms present in vent communities was investigated using specific query (marker) genes that code for proteins known to mediate the assimilation of inorganic C, electron transfer, and/or stress response (Table 4 ). Nearly, all phylotypes identified using different functional genes were consistent with those determined using phylogenetic analysis of 16S rRNA genes (e.g., Figures 3 , 4 , Tables 2 , 3 ). Consequently, a consistent picture emerges regarding the functional attributes of major population types identified in IP and WT (Table 4 ). The low fraction of assembled sequence obtained from the MB vent sediments precluded confident assignment, and the majority of genes identified were less than 25–30% of their full length (not shown). Table 4 Summary of functional genes a (and their phylogenetic identity b ) related to key geochemical processes, which were identified in assembled metagenome sequence of three thermal vent microbial communities from Yellowstone Lake, WY . Process/Pathway Marker genes a Inflated Plain (IP-348S) Inflated Plain (IP-359S) West Thumb (WT-369S) FIXATION Of CO 2 ATP citrate lyase aclB Sk, Sy Sk, Sy Citryl coA lyase ccl Sy Sy Sy Citryl coA synthetase ccsA Tu Ce, Sk, Sy At, Sy, Td Acetyl-coA carboxylase accA Ce, Sy, Tp Acid, Ce, Sk, Sy, Thio Ma, Td OXIDATION REDUCTION Hydrogen oxidation hynS, hynL Sy, Tp, Tu, Sy, Tp Td Elemental S oxidation hdrA/hdrB Sy Sy Sy, Td Sulfide oxidation sqr Sy, Sk, Sy, Thio Td, Sulfur transferase rdh Sk, Sy Sy, Sk Nl, Sy, Td Sulfur oxidation soxBCDY Sy, Ce, Sy, Thio Sy, Td Methane oxidation pmoABC Ms Formate oxidation fdh As, Ce Acid, Ce Ma Oxygen reduction, Heme Cu Oxidases cbb3 Sy Rf, Sk, Sy, Thio Sy, Td Oxygen reduction, bd-ubiquinol type cydA As, Py, Sy, Tp, Tu Py, Tp Thdes Sulfur reduction psrA/sreA c Py, Sy, Tu Py Sulfate reduction dsrAB Des, Td Nitric oxide reduction norB Py, Tu Py Nitrate reduction narG Sk STRESS RESPONSE Arsenite efflux-detoxification arsB As, Py, Sy, Tu Sk, Sy Sy Arsenate reduction-detoxification arsC Ce, Sy Ce, Sy Mercuric reductase merA Thio Heavy-metal ATPases znta Sy, Thio Ma, Sy Superoxide dismutase sodA As, Py, Tu Acid, Py, Thio Mp Hydroperoxide reductase (peroxiredoxin) perox As, Ce, Sy, Tp, Tu As, Ce, Sk, Sy, Tp Ma, Sy, Ta, Td, Thio Desulfoferrodoxin (superoxide reductase) sorA Ce, Tp Ce, Tp Rubredoxin rub Ce, Sy Acid, Ce, Sy Thdes Motility flaB Sy, Thio Sy, Thio Fp, Sy a Functional genes that code for proteins with high specificity for possible pathway; no genes were found for nitrification (amoA), denitrification (e.g., nirK, nirS, nosZ), methanogenesis (mcrA), thiosulfate oxidase (tqoAB), or arsenite oxidation (aroA = aioA); a ferric reductase from an Acidovorax sp. population was observed in WT. b Population Types (closest relatives): Acid, Acidovorax sp.; As, Acidilobus saccharvorans; At, Anaerolinea thermophila; Ce, Caldisericum exile; Des, Desulfobacterium sp.; Fp, Fervidobacterium pennivorans; Ma, Methylomicrobium alcaliphilum; Ms, Methylothermus subterraneus; Mp, Mucilaginibacter paludis; Py, Pyrobaculum sp.; Tu, Thermoproteus uzoniensis; Thio, Thiovirga sulfuryoxidans; Sy, Sulfurihydrogenibium sp.; Sk, Sulfuricurvum kujiense; Tp, Thermofilum pendens; Td, Thiobacillus denitrificans; Ta, Thermocrinis albus; Nl, Nitrosoarchaeum limnia; Thiom, Thiomonas sp.; Thdes, Thermodesulfobacteria. c includes unclassified DMSO proteins that may be related to sulfur and/or arsenic reduction in the Thermoproteales (Jay et al., 2015 ). Metabolic evidence for the fixation of carbon dioxide (CO 2 ) via the reductive TCA cycle (e.g., ATP citrate lyase; Takacs-Vesbach et al., 2013 ) was identified in all streamer communities, and was especially evident in Sulfurihydrogenibium (Aquificales) populations (Table 4 ). Copies of acetyl-CoA carboxylase ( accA ) were noted in several bacterial phylotypes as well as a Thermoproteales population in IP 348S. In bacteria, acetyl-coA carboxylase is required for the synthesis of fatty acids. Consequently, the phylogenetic identity of these genes is essentially consistent with the major bacterial phylotypes present across the 3 streamer communities. In the Archaea , acetyl-CoA carboxylase is involved in the 4-hydroxybutyrate/3-hydroxyproprionate CO 2 fixation cycle (or decarboxylase version) (Berg et al., 2007 ); however, this gene was only observed in the Thermofilum pendens -like population present in one of the IP streamer communities (348S), and other key marker genes for the 4-HB/3-HP pathway were not observed (Berg et al., 2007 , 2010 ). Consequently, the sequence data suggest that the primary mechanism of CO 2 fixation in these communities occurs via the reductive-TCA cycle (Beh et al., 1993 ; Hügler et al., 2007 ), and supports measurements of dark CO 2 fixation rates obtained in a prior study (Yang et al., 2011 ). Genes coding for proteins known to be important in the oxidation of reduced sulfur species were observed in these communities, and were most-closely related to the dominant bacterial populations present including Sulfurihydrogenibium, Sulfuricurvum, Thiovirga, Thiobacillus , and Caldisericum spp. (Table 4 ). Specifically, hdrAB genes indicative of a S oxidation pathway (Friedrich et al., 2005 ) were found in Sulfurihydrogenibium sequences, as has been observed in terrestrial sites of YNP (Takacs-Vesbach et al., 2013 ). Several other key marker genes and pathways for S oxidation ( sqr, sox ) were identified as Sulfuricurvum, Thiobacillus , and Thiovirga spp., as well as Sulfurihydrogenibium -like (Table 4 ). Group I Ni-Fe hydrogenases, indicative of H 2 uptake and oxidation (Viginais and Billoud, 2007 ), were found in Sulfurihydrogenibium, Thermofilum, Thermoproteus , and Thiobacillus -like assemblies (Table 4 ). The hydrogenases present in the Sulfurihydrogenibium -like sequence assemblies are most closely related to other Aquificales genera, because the only known Sulfurihydrogenibium sp. to contain a Group 1 Ni-Fe hydrogenase is S. azoricus (Aguiar et al., 2004 ; Reysenbach et al., 2009 ). To date, the Sulfurihydrogenibium -like populations characterized in terrestrial sites of YNP do not contain Group I Ni-Fe hydrogenases (Inskeep et al., 2010 ; Hamamura et al., 2013 ; Takacs-Vesbach et al., 2013 ). The higher concentrations of H 2 (aq) (>4 μM) at IP vent sites correlates with the presence of hydrogenases in Sulfurihydrogenibium -like sequences found in two replicate streamer communities (348S, 359S). A near-complete methane oxidation pathway (particulate methane monooxygenase subunits ABC) was identified in the streamers from WT (369S) (with the exception of formaldehyde dehydrogenase). The pmoABC genes were most closely related to genes from the gamma-proteobacterium Methylothermus subterraneus (95% nt identity for pmoA ) (Tsubota et al., 2005 ; Hirayama et al., 2011 ). Bacterial populations (similar to M. subterraneus and M. thermalis ) were identified as major taxa in WT streamers (pH ~ 6.1) in both 2007 and 2008 ( n = 3) (Table 2 ). Moreover, no pmoABC genes were identified in other vent sites. The pmoCAB operon architecture (Ward et al., 2004 ) was not recovered from the metagenome assembly, and a definitive pathway of CO 2 fixation via formaldehyde assimilation could not be determined, as the key gene for the ribulose monophosphate pathway (3-hexulose-6-phosphate synthase) was not identified. To date, pmoABC genes have not been observed in metagenomes from numerous terrestrial sites in YNP (Inskeep et al., 2010 , 2013a ; Swingley et al., 2012 ). Moreover, methanotrophs and/or methylotrophs have not been observed as dominant population types in terrestrial thermal habitats characterized to date, despite fairly high concentrations of CH 4 (aq) in some locations (e.g., 1–2 μM; Inskeep et al., 2005 , 2013a ). Concentrations of CH 4 (aq) measured in vent sites at IP, WT, and MB ranged from 5 to 30 μM (Table 1 ); however, WT (i.e., pH ~ 6; T ~ 60°C, lower sulfide) was the only site to exhibit abundant methanotrophic population(s). The lower pH values and higher sulfide of vents at IP and MB (Table 1 ) may preclude methanotrophic populations, as these conditions are not optimum for the oxidation of CH 4 using O 2 as an electron acceptor (Tsubota et al., 2005 ). Oxygen is an important electron acceptor in thermal vent communities of Yellowstone Lake as evinced by the presence of Type C (cbb3) heme Cu oxidases in many of the dominant bacterial population types, including Sulfurihydrogenibium, Sulfuricurvum, Rhodoferax, Thiomonas , and Thiobacillus -like populations (Table 4 ). These types of heme Cu oxidases have been shown to exhibit low K m values for O 2 , and are often found in hypoxic environments (García-Horsman et al., 1994 ; Jünemann, 1997 ; Borisov et al., 2011 ). Ubiquinol oxidases (e.g., cydA ) were also observed in several archaeal populations present in the highly sulfidic sites at IP (e.g., Desulfurococcales and Thermoproteales; Jay et al., 2014 , 2015 ) as well as in the Thermodesulfobacteria at WT. These oxidases are common in hypoxic environments and may function in respiration or as O 2 scavenging proteins (Borisov et al., 2011 ). Other electron acceptors important for specific members of these communities may include elemental S, arsenate, nitrate, and sulfate (Table 4 ). Novel DMSO molybdopterins (tabulated as psrA/sreA ) related to Sulfurihydrogenibium and Thermoproteales populations in the IP streamers may play a role in the reduction of elemental sulfur and/or arsenate (Jay et al., 2015 ), and these metabolisms would be expected within the S-rich streamer fabric (Figure S2 ). The only evidence of dissimilatory nitrate reduction ( narG ) was found in the Sulfuricurvum population from IP. The role of norB genes present in several Thermoproteales populations is not fully understood (NorB may also exhibit activity as an oxygen reductase), in part because no evidence for a complete denitrification pathway has been documented in this group of organisms (Jay et al., 2015 ). The only evidence of sulfate reduction ( dsrAB ) was associated with nonthermophilic populations at WT (i.e., Thiobacillus, Desulfobacteria )."
} | 10,355 |
40279424 | PMC12024680 | pmc | 6,020 | {
"abstract": "Increasing efforts have been devoted to developing biobased and biodegradable plastics and composites from lignocellulosic biomass. Current bioplastic production entails multiple challenging steps including monomer production from biomass as well as polymer synthesis and modification. Here, we report a practical recombination strategy to transform agricultural residues into moldable cellulose-reinforced lignin (CRL) composites. The strategy involves deconstruction of biomass particles followed by thermo-compression molding of cellulose fibers and lignin mixtures. The resulting CRL composites demonstrated excellent mechanical and thermal properties as well as water, abrasive, and flame resistance. Mechanistic studies reveal that small particle size, removal of water-soluble fractions, as well as reservation of lignin and its cross-linking reactivity have considerably positive effects on preparation of high-quality composite items. These insights offer a versatile strategy for transforming various types of low-grade biomass, such as corn stover, into eco-friendly and potentially biodegradable or compostable composites that can serve as sustainable alternatives to traditional duroplast materials.",
"introduction": "INTRODUCTION Increasing concerns about plastic and microplastic pollution have raised worldwide efforts to develop biodegradable plastics and composites from renewable lignocellulosic biomass such as corn stover (CS) and wood ( 1 – 3 ). Current biomass-to-bioplastics conversion processes involve hydrolysis of cellulose to sugars, conversion of sugars to monomers, as well as synthesis of polymer from monomers ( 4 – 6 ). Before practical uses, polymers also need to be mixed with fillers or reinforcement materials such as fiber and powder materials to form composites to improve the properties of polymers and reduce the cost of bioplastics ( 7 – 9 ). Alternatively, bioplastics (e.g., cellulose acetate, lignin-based epoxy, and lignin-phenol-formaldehyde resin) could also be produced from lignocellulose components with pretreatment ( 10 ) and chemical modification ( 11 – 13 ) to improve the plasticity and reactivity of biomass components. However, the above approaches for bioplastics fabrication focus on conversion of a single component and require multiple challenging steps ( 14 ). Alternatively, biomass particles can also serve as reinforcement materials or fillers of thermoplastics (e.g., polyethylene, polylactic acid, and polyethylene terephthalate) and thermoset resins (e.g., epoxy and unsaturated polyester resins) for wood-based composites preparation ( 15 – 17 ). During preparation of biomass particle–reinforced polymeric composites, additional surface treatments of biomass particles (e.g., acetylation and silylation) are required to enhance interfacial compatibility between hydrophilic particles and hydrophobic polymer matrixes ( 17 , 18 ). However, the uses of petro-based polymeric matrices such as phenol-formaldehyde resin ( 19 , 20 ) and epoxy resin ( 21 – 23 ) that are often not chemically recyclable pose challenges to the environment and human health. Consequently, the development of straightforward and practical strategies to transform low-quality agricultural residues into high-quality biobased composite materials is both urgent and critically important. Such advancements would provide sustainable alternatives to conventional polymeric composites in various everyday applications, thereby benefiting human society and the environment. Inspired by our previous study that lignin could be used as an adhesive for binding wood ( 24 ), we believed that lignocellulosic biomass could be directly recombined into composites with cellulose fibers acting as reinforcement and lignin acting as a thermoset polymer matrix, respectively. However, the complex cross-linking among major components (cellulose, hemicelluloses, and lignin) of lignocellulosic biomass leads to an elevated softening temperature close to its degradation temperature (>200°C) ( 25 , 26 ). The degradation of biomass components occurred before the occurrence of extensive thermal softening of biomass particles. Therefore, rigid and hydrophilic biomass materials exhibit lower plasticity and self-bonding properties than conventional plastics and are hard to be directly reformed into high-performance and water-resistant plastic-like structural materials. To achieve our goal of processing biomass into moldable composite materials, the following four aspects were carefully considered during our investigation. First, small biomass particles rather than long fibers or large particles were used to improve interfacial interaction. Second, biomass particles were deconstructed to remove thermo-unstable extractives and hydrophilic hemicelluloses to increase the thermo-stability and water resistance of biomass materials. Third, lignin, which served as a hydrophobic polymeric matrix, was depolymerized and reserved during biomass deconstruction to wet and bind cellulose. Fourth, the cross-linking reactivity of lignin was maximally saved during extractives and hemicelluloses removals. On the basis of this conception, we successfully prepared previously unknown cellulose-reinforced lignin (CRL) composites from a wide variety of low-quality lignocellulosic biomass via a cell wall recombination strategy. The cell wall recombination process is shown in fig. S1. First, acetal-protection (AP) treatment was used for simultaneous deconstruction and in situ modification of fine biomass particles. During AP treatment, cellulose and lignin contents were improved via removing undesired water-soluble fractions. Subsequent thermo-compression molding promoted thermal softening and self–cross-linking of lignin to tightly bond cellulose together. In this strategy, high-quality items with designable shapes, various colors, and performances comparable to natural wood and structural timber products (e.g., glued-laminated timber and laminated veneer lumber) are prepared from agricultural residues ( Fig. 1, A and B , and table S1). These completely biobased items after use showed the potential to be recovered as cheap raw materials for biorefining ( Fig. 1C ). Fig. 1. Preparation of CRL composites from agricultural residues via cell wall recombination. ( A ) Photographs of CRL composites with designable shapes and various colors prepared from agricultural residues. ( B ) Comparison of MOR between CRL composites (up to 55.2 ± 2.1 MPa) and natural woods (19.6 to 107.8 MPa) and wood-derived products (15.2 to 86.2 MPa) from literatures ( 38 – 41 ). ( C ) Proposed pathways for recovering CRL composites as raw materials for biorefining. The photograph showed a cellulose-lignin slurry resultant from CRL composites after 6-hour immersion at room temperature in a 0.25 M NaOH aqueous solution. Photo credits: All photos presented in the article were taken by Z.G.",
"discussion": "DISCUSSION In conclusion, we have developed a simple and practical strategy to transform lignocellulosic biomass, such as agricultural residues and wood, into high-quality biocomposites as sustainable alternatives to synthetic plastics. Our findings highlight that the complete deconstruction of biomass, the removal of hemicelluloses and extractives to enhance lignin content, and the preservation of lignin along with its cross-linking reactivity are all critical factors for the efficient fabrication of CRL composites under industrially feasible thermo-compression conditions. By using mild thermo-compression techniques and light-colored moldable materials (e.g., AP-CS), we were able to produce light-colored and customizable composite materials. Furthermore, these composites exhibit great potential for recovery through composting or as cost-effective raw materials for biorefining processes. While this straightforward and scalable strategy presents a promising approach to addressing the environmental challenges associated with excessive use of nondegradable petroleum-based synthetic plastics, CRL composites can only serve as a partial substitute for traditional thermoplastics, thermosets, or their composites in specific application areas. This limited applicability stems from several inherent constraints, including relatively low elongation at break, susceptibility to water and gas permeability, and challenges in molding processes."
} | 2,085 |
35278217 | PMC9544845 | pmc | 6,023 | {
"abstract": "Abstract \n The spatial distribution of animals in a landscape depends mainly on the distribution of resources. Resource availability is often facilitated by other species and can positively influence local species diversity and affect community structure. Species that significantly change resource availability are often termed ecosystem engineers. Identifying these species is important, but predicting where they have large or small impacts is a key challenge that will enhance the usefulness of the ecosystem engineering concept. In harsh and stressful environments, the stress gradient hypothesis predicts that community structure and function will be increasingly influenced by facilitative interactions. To test this hypothesis, we investigate how the ecosystem engineering role and importance of sociable weavers Philetairus socius varies across a spatial gradient of harshness, for which aridity served as a proxy. These birds build large colonies that are home to hundreds of weavers and host a wide range of avian and non‐avian heterospecifics. We investigated the use of weaver colonies on multiple taxa (invertebrates, reptiles, birds and mammals) at multiple sites across a >1,000 km aridity gradient. We show that sociable weaver colonies create localized biodiversity hotspots across their range. Furthermore, trees containing sociable weaver colonies maintained localized animal diversity at sites with lower rainfall, an effect not as pronounced at sites with higher rainfall. Our results were consistent with predictions of the stress gradient hypothesis, and we provide one of the first tests of this hypothesis in terrestrial animal communities. Facilitation and amelioration by ecosystem engineers may mitigate some of the extreme impacts of environmental harshness.",
"conclusion": "5 CONCLUSIONS Our findings add to the increasing literature that ecosystem engineering is an important biological interaction that can relieve stress in harsh environments (Coggan et al., 2018 ; Hastings et al., 2007 ; Romero et al., 2015 ). We also show the key role of sociable weavers as allogenic engineers in structuring animal communities throughout their range. Furthermore, associations with terrestrial vertebrates and birds to weaver colonies increased in more harsh environments supporting predictions of the SGH, suggesting that colonies may mitigate some of the additional stresses experienced by associated wildlife as climate change advances.",
"introduction": "1 INTRODUCTION The spatial distribution of animals within a landscape is largely determined by the availability of resources (Hunter et al., 2012 ; McIntyre & Wiens, 1999 ), which can be concentrated in specific locations (Parrish & Edelstein‐Keshet, 1999 ). Resource availability can be facilitated by other species, and this can positively affect local species diversity and impact community structure (Soliveres et al., 2015 ). Species that considerably alter resource availability in an environment are known as ecosystem engineers (Jones et al., 1994 ). This concept is not without criticism (Reichman & Seabloom, 2002 ), but it is agreed that significant value can be derived by identifying those ‘engineers’ that disproportionately influence resource availability and have the greatest abiotic and biotic impacts on their environment (Coggan et al., 2018 ; Crain & Bertness, 2006 ; Romero et al., 2015 ). However, the impacts of ecosystem engineers have mainly been carried out across small spatial scales, limiting our understanding of how spatial context may alter impacts (Coggan et al., 2018 ). The stress gradient hypothesis (SGH) predicts that the significance of facilitative interactions will increase in communities in harsher environments (Bertness & Callaway, 1994 ). This is supported by empirical evidence demonstrating greater associative and positive between‐species impacts with increasing environmental stress, often facilitated by identified ecosystem engineers (He et al., 2013 ). To date, studies into the SGH have almost been exclusively tested in plant communities; however, recently, ecologists started to apply these ideas to animal communities (Dangles et al., 2018 ; García‐Navas et al., 2021 ; Lowney & Thomson, 2021 ). Moreover, studies across broad spatial scales are challenging to replicate but may demonstrate the importance of engineering species to different communities and in different contexts. Environmental conditions (i.e. aridity, altitude, salinity) will likely vary significantly across an engineering species' distribution (Coggan et al., 2016 ; Erpenbach et al., 2012 ). Therefore, monitoring an engineer's impact over large‐scale spatial ecological gradients would enable a greater understanding of how engineers may facilitate or mitigate condition in environments differing in harshness. Species interactions are likely a key factor as communities respond to climate change (Alexander et al., 2015 ; Harrington et al., 1999 ; Suttle et al., 2007 ). In arid environments, climate change will predominantly cause increasing frequencies and duration of hot weather or drought events (Akoon et al., 2011 ; Meehl & Tebaldi, 2004 ), and a reduction in rainfall (Osman et al., 2017 ; Ouhamdouch & Bahir, 2017 ), conditions that will make these environments harsher to most species (Erasmus et al., 2002 ; Isaac, 2009 ). Altered species interactions due to an environment becoming too harsh may lead to a loss of certain species from communities, long before species‐specific temperature thresholds are reached. Ecosystem engineers join the abiotic and trophic aspects of communities via their interaction networks (Sanders et al., 2014 ). Engineered structures that provide thermal refuges may be crucial under increasingly higher temperatures (Coggan et al., 2018 ), resulting in increased use of these structures. Burrowing by animals may provide these refuges but also alters the soil properties that directly influence local plant community composition (Bancroft et al., 2008 ; Whitford & Kay, 1999 ). Therefore, by influencing plant biomass they have the potential to provide resources in periods of low plant productivity. Bird nests have the potential to provide resources for many different species as they come in different shapes and forms, with large communal nests providing resources for species that gravitate towards these structures, and nests burrowed underground that alter vegetation structural complexity and vertebrate fauna (Bancroft et al., 2008 ; Delhey, 2018 ; Lowney & Thomson, 2021 ; Mainwaring et al., 2015 ; Natusch et al., 2016 ). Yet a recent review revealed that very few studies have investigated birds as terrestrial ecosystem engineers, instead a considerable bias towards invertebrates and mammals was observed (Coggan et al., 2018 ). Animals living in arid habitats regularly face harsh conditions. Maximum and minimum temperatures can exceed the upper and lower thresholds of many species and precipitation is unpredictable, resulting in fluctuations between scarce or plentiful vegetation cover (Hillel & Tadmor, 1962 ; Rosenzweig, 1968 ), which determines the availability of resources to other species further up the food web (Polis, 1991 ). The impact of any engineer may change depending on these environmental contexts. Using a spatial aridity gradient allows for comparison of species interactions with ecosystem engineers as environmental stress increases. This approach may enhance predictions of how animal community structure and species interactions may change as benign sites become harsher and how engineers could mitigate stress. Our aim is to determine the role of an avian ecosystem engineer on animal species diversity, and we use this system to test how its impacts may change across spatial gradients of environmental harshness. Our focal species is the sociable weaver Philetairus socius (henceforth weaver). These small passerines construct large nest colonies and are endemic to the semi‐arid and arid areas of the western parts of southern Africa (Maclean, 1973 ; Mendelsohn & Anderson, 1997 ). Colonies can contain hundreds of nesting chambers and are inhabited and maintained year‐round, meaning that some colonies can remain active in the environment for over a century (Maclean, 1973 ). In addition, colonies are also dynamic and can increase in size from year to year or completely collapse. Larger colonies may host hundreds of individual birds and nest chambers provide insulation, a crucial resource in arid environments, for its occupants (Lowney et al., 2020 ). Soils directly below colonies have particularly increased nutrient levels (Prayag et al., 2020 ) and this could result in direct effects on the local vegetation and animals. Weaver colonies have been shown to act as a resource to multiple species within the environment (Bolopo et al., 2019 ; Lowney & Charlton, 2017 ; Maclean, 1970 ; Rehn, 1965 ; Rymer et al., 2014 ) and maintain this impact throughout the year (Lowney & Thomson, 2021 ). Their facilitative role across their distribution remains unknown (Figure 1 ). FIGURE 1 A sociable weaver colony at one of the study sites. This colony was at Tswalu Kalahari Reserve and contained 167 chambers We undertook a meta‐replicated study investigating weaver colony use by local animal communities at sites across a >1,000 km gradient of the weaver's distribution. The eight sites selected differed in aridity and represent a ‘harshness gradient’ that allowed us to test the predictions of the SGH. We hypothesized that because weaver colonies ameliorate harsh abiotic conditions and provide biotic resources, that as conditions became harsher these mechanisms would increasingly buffer animal communities. We expected to observe greater animal diversity of multiple taxa at trees containing a weaver colony compared with control trees without a colony across all sites. Most species that use weaver colonies are not obligate associates and can exhibit behavioural plasticity in their use of weaver colonies. We hypothesize that because invertebrate abundance is influenced by organic matter (Noy‐Meir, 1985 ) and this collects below colonies (Prayag et al., 2020 ), that a greater abundance of invertebrates will be observed at colony trees than the control trees. We also hypothesize that due to thermal insulation against hot and cold temperatures that colonies provide (Lowney et al., 2020 ) and increased resources in terms of invertebrate abundance, small‐ to medium‐sized birds and reptiles will associate with colony trees. We hypothesize that use of colonies for shade and territory marking (Lowney & Thomson, 2021 ) will increase the associations of large mammals, while herbivores are likely to forage more at colony trees due to the nutrient‐rich vegetation and increased foliar biomass (Prayag et al., 2020 ). Therefore, we expect increased number of large vertebrates at colony trees. The SGH predicts that positive interactions should be more frequent in communities under high physical stress (Bertness & Callaway, 1994 ). Therefore, we hypothesize that the relative number of animals that interact with colony trees would increase at sites with lower rainfall and normalized difference vegetation index (NDVI) values, and we predicted that the use of colonies would increase at sites with lower rainfall and lower plant productivity.",
"discussion": "4 DISCUSSION Our study demonstrates that weaver colonies enhance local diversity across their range and that these strong associations are a replicated and consistent feature of these structures. For all taxa sampled (invertebrates, birds, reptiles and mammals), we found increased numbers of individuals associated with camel thorn trees hosting weaver colonies. Furthermore, we observed increased species richness in invertebrates and roosting birds, and increased species diversity in invertebrates and vertebrates associated with camel thorn trees hosting weaver colonies. Likely through a variety of mechanisms, weaver colonies create habitat and enhance resources to surrounding animal communities, serving as an ecological engineer. Importantly, our results suggest that weaver colonies were associated with increased relative numbers of animal events and diversity especially at the more arid sites for both birds and terrestrial invertebrates. Camel thorn trees containing weaver colonies maintained higher biodiversity and animal associations despite increasing aridity at sites, in contrast to non‐colony trees where overall animal diversity and associations decreased as aridity increased. This provides support for a prediction of the SGH, where facilitation by these ecosystem engineers becomes increasingly important in animal communities as harshness of the environment increases. 4.1 Abundance of invertebrates Both terrestrial and aerial invertebrates demonstrated a strong association with colony trees, which was maintained at the more arid sites. Weaver colonies clearly provide an environment and resources suitable for invertebrate diversity. Additionally, aerial invertebrates saw an increase in the number of individuals, species abundance and species richness at wetter sites. Factors key to insects in arid environments are temperature, water availability and the presence of organic matter (Noy‐Meir, 1985 ). Organic matter collects underneath colonies in the form of fallen nest material and with potentially hundreds of avian residents, faeces deposited can form substantial faecal mats that would be an essential source of organic matter in these environments (Prayag et al., 2020 ). The more arid an environment, the greater the importance of this source of organic matter would be, and this would explain the difference between terrestrial invertebrate interactions with colony and non‐colony trees across the environmental gradient. Invertebrates form an important resource for other taxa too, therefore their strong association may feedback into further positive associations of other taxa with colony trees. Not only does this add further support for the importance of birds as ecosystem engineers, but this also demonstrates that a particular species can become more important to the local animal community as environmental harshness increases, supporting the SGH. Furthermore, this is evidence that the SGH can be tested across animal communities and not confined to single pairwise species interactions. 4.2 Abundance of reptiles Colony presence increased the abundance of reptiles. Our findings support previous research that showed reptiles associate with weaver colonies (Rymer et al., 2014 ). However, we did not see variation between the differences in colony and non‐colony trees across the aridity gradient. The main factors that influence the abundance of reptiles are food availability and the ability to thermoregulate (Corbalán et al., 2013 ), and colonies provide shelter and resources. The two tree skink species observed ( Trachylepis spp.) are insectivores, and invertebrate abundance is higher around colony trees. Therefore, colonies provide the primary resources needed by reptiles in arid environments and explain why colony trees have greater interactions with reptiles than non‐colony trees across all sites. The increase in numbers at wetter sites is likely driven by productivity leading to an increase in prey. Given that only two reptile species were observed, it may be more applicable to install pitfall traps with drift fences around colony and non‐colony trees to determine the response of this taxa more reliably (Larsen, 2016 ). 4.3 Abundance of avian species and roosting birds Birds showed an association for colony trees in more arid sites. In harsh environments, small‐ to medium‐sized birds avoid high temperatures by seeking refuge (Willmer et al., 2009 ). Weaver colonies should provide permanent shade and the chambers have been shown to buffer against harsh temperatures (Lowney et al., 2020 ; Lowney & Thomson, 2021 ). All the birds observed during point counts were small‐ to medium‐sized birds, suggesting that this kind of facilitation would explain the differences observed between the interactions with colony and non‐colony trees across the environmental gradient. Additionally, insects are an essential nutrition and water source for many bird species. Therefore, strong invertebrate associations found at weaver colonies may explain why more birds associate with colony trees in areas of low rainfall. Our results also showed the importance of colonies for roosting birds; 51% of colonies hosted heterospecifics on the single sampled night. Larger colonies were more likely to attract heterospecifics and had a greater species richness of roosting birds. More chambers mean more available buffered ‘resources’ for other species to use as roosting sites. In addition, roosting with hundreds of other heterospecifics may reduce the risk of predation via the dilution effect (Beauchamp, 1999 ). Our quantitative data, together with anecdotal data, show weaver colonies are important to several bird species; rosy‐faced lovebird Agapornis roseicollis breed in weaver colonies (Ndithia et al., 2007 ), while other raptor species have been observed using weaver colonies as platforms to nest on (Oschadleus, 2019 ). 4.4 Abundance of vertebrates Mammals associated with colony over non‐colony trees, but we did not detect that the relative importance of colony trees increased across the aridity gradient. This lack of impact across the gradient in vertebrates could be explained by how engineers provide facilitation towards different taxa. Many of the terrestrial and arboreal mammals captured by camera traps are species that would need to reduce their metabolic heat production by reducing activity during the hottest parts of the day (Willmer et al., 2009 ). We have previously found that mammals use weaver colonies for shade (Lowney & Thomson, 2021 ). However, this study did not include the hottest time of the year and as a result this behaviour was not frequently observed. We speculate that during summer‐time sampling, the increased importance of shade and moisture as a resource would increase the relative importance of colony trees in drier and hotter sites. Furthermore, smaller sample sizes relative to invertebrate and avian data may result in a false negative result. We suggest further studies with a recommendation to increase sample sizes, different seasons and possibly over more extended periods (e.g. camera trapping for a minimum of 2 weeks, Larsen, 2016 ). Smaller sample sizes also meant that we combined the terrestrial and arboreal data, as a result these were not tested independently. 4.5 Colony use across a spatial gradient The positive diversity impacts of colonies were most pronounced on birds and terrestrial invertebrates at sites with low rainfall. Colony presence increased the number of individuals and species richness at low rainfall sites relative to non‐colony trees; however, the relative difference almost disappeared when environmental conditions became less severe. Thus, the net effect of colonies changed from positive associations of animal communities in the more stressful sites to almost neutral at sites that were benign. This study provides empirical support for the SGH and complements recent research using terrestrial animal communities that found evidence of increased importance of associative or facilitative interactions in harsh environments (Bell & Cuddington, 2019 ; Dangles et al., 2018 ; García‐Navas et al., 2021 ). Additionally, by demonstrating that some taxa associate strongly with colony trees in more arid climates, we provide support that facilitation by weaver colonies increases the realized niches of certain species (Armas et al., 2011 ; He & Bertness, 2014 ). Furthermore, only one other study has extended this hypothesis to free‐ranging animals (García‐Navas et al., 2021 ), as many of the previous studies were tested under laboratory conditions (Bell & Cuddington, 2019 ; Dangles et al., 2018 ). Temporal variations in climatic conditions may also play a part in understanding the facilitative role of weaver colonies. This we did not test, but a previous study failed to demonstrate any variation in the use of weaver colonies by heterospecifics across a calendar year (Lowney & Thomson, 2021 ). However, due to the unpredictability of weather events throughout the weaver's range, this time frame may still be too short to observe variation across a temporal gradient (Lowney & Thomson, 2021 ). Our site visits were not carried out concurrently, and therefore may have some effects on our results. However, our study design of using paired trees should minimize these impacts. It is likely that such facilitation plays an important role, allowing multiple taxa to persist in environments that may otherwise be too harsh. Weaver colonies provide different resources for different taxa and, in turn, create ecological hotspots around colony trees. Consequently, colony presence at more stressful sites likely causes more isolated hotspots of life. Many species in harsh habitats have adaptations that allow them to survive with the extreme stresses associated with these environments (Bennett et al., 1984 ; Schmidt‐Nielsen et al., 1969 ; Williams & Tieleman, 2005 ). However, many of the species sampled use colonies as thermal refuges (Lowney & Thomson, 2021 ). Therefore, our results suggest that, in a landscape that will become increasingly harsh as climate change advances (Akoon et al., 2011 ), these colonies will become critical ecosystem components that will buffer some of the harsh environmental conditions and allow some species to persist despite these tougher conditions. To conserve biodiversity and reduce the impacts of climate change, it is important to understand how facilitation within animal communities is undertaken and how the processes and interactions within ecosystems are maintained (Coggan et al., 2018 ). If external temperatures exceed a threshold that colonies can no‐longer sufficiently buffer against, this will have severe consequences for animal communities in these areas. We must understand the role that engineers will play on maintaining ecosystems in an environment changing due to human‐induced climate change and that this should be a priority for future research."
} | 5,599 |
37877659 | PMC10505944 | pmc | 6,024 | {
"abstract": "Abstract The light conditions are of utmost importance in any microalgae production process especially involving artificial illumination. This also applies to a chrysolaminarin (soluble 1,3‐β‐glucan) production process using the diatom Phaeodactylum tricornutum . Here we examine the influence of the amount of light per gram biomass (specific light availability) and the influence of two different biomass densities (at the same amount of light per gram biomass) on the accumulation of the storage product chrysolaminarin during nitrogen depletion in artificially illuminated flat‐panel airlift photobioreactors. Besides chrysolaminarin, other compounds (fucoxanthin, fatty acids used for energy storage [C16 fatty acids], and eicosapentaenoic acid) are regarded as well. Our results show that the time course of C‐allocation between chrysolaminarin and fatty acids, serving as storage compounds, is influenced by specific light availability and cell concentration. Furthermore, our findings demonstrate that with increasing specific light availability, the maximal chrysolaminarin content increases. However, this effect is limited. Beyond a certain specific light availability (here: 5 µmol photons g DW \n −1 s −1 ) the maximal chrysolaminarin content no longer increases, but the rate of increase becomes faster. Furthermore, the conversion of light to chrysolaminarin is best at the beginning of nitrogen depletion. Additionally, our results show that a high biomass concentration has a negative effect on the maximal chrysolaminarin content, most likely due to the occurring self‐shading effects.",
"conclusion": "4 CONCLUSION In conclusion, light is an important factor in a possible microalgae production process involving chrysolaminarin production. Our results showed that for a chrysolaminarin production process, only a N‐depletion phase of up to 4 days is needed and sensible because the maximal amount of chrysolaminarin was accumulated in the first days of N‐depletion. The depletion phase can be even shortened with higher I \n spec . Although a higher I \n spec fastened chrysolaminarin accumulation, it did not necessarily increase the maximal chrysolaminarin content. Above a certain I \n spec (here 5 µmol photons g DW \n −1 s −1 ) the maximal chrysolaminarin content did not increase further. A higher initial biomass concentration led to a lower maximal chrysolaminarin content, indicating that self‐shading effects were of relevance here.",
"introduction": "1 INTRODUCTION High‐value compounds from microalgae, such as glucans, pigments, and fatty acids, have continued to attract interest in recent years, for example for the application in food, feed, and cosmetics (Gora et al., 2022 ; Lourenço‐Lopes et al., 2021 ; Neumann, Derwenskus, et al., 2018 ; Neumann, Louis, et al., 2018 ; Reis et al., 2021 ; Stiefvatter et al., 2021 ). For the production of microalgae‐derived components, the cultivation conditions are of outstanding importance (Derwenskus, 2020 ). During the phototrophic cultivation of microalgae, light serves as the only energy source for biomass formation and further the formation of desired compounds. Therefore, the light conditions have a great impact on autotrophic microalgae production processes. During phototrophic cultivation in closed photobioreactors, the amount of light available to a microalgae culture can be directly linked to its biomass productivity (Derwenskus, 2020 ; Holdmann et al., 2018 ). The light impinging on the surface of photobioreactors can be controlled. For example by shading when using natural light during outdoor cultivation or by dimming the light source when using artificially illuminated photobioreactors. In any case, the light available to every gram of biomass—the specific light availability ( I \n spec )—is of special interest for the process (Holdmann et al., 2018 ). I \n spec describes the ratio between the photon flux density (PFD) (µmol photons m −2 s −1 ) on the surface of the photobioreactor to the total amount of biomass in the reactor volume (Holdmann et al., 2018 ). It not only has an impact on biomass productivity but also on the biomass composition, for example, on the accumulation of fucoxanthin (FX) in Phaeodactylum tricornutum (Derwenskus, 2020 ). Although already considered in the calculation for I \n spec , the culture density has a crucial impact on the light conditions in photobioreactors. With rising biomass density and despite mixing, self‐shading effects occur in microalgae cultures, affecting biomass productivity (Holdmann et al., 2018 ). In modern biotechnological processes, efforts should be made to use resources as efficiently as possible and not to waste them needlessly. In contrast to the outdoor production of microalgae biomass, artificially illuminated processes require electricity for light generation. Therefore, in order not to waste resources like electricity, it is of particular importance in artificially‐illuminated processes how efficiently light is converted into biomass or a desired product (light yield). Moreover, in production processes using artificial illumination, the energy costs for illumination are a major part of the operating costs of the process (Derwenskus, Weickert, et al., 2020 ). Microalgae contain a multitude of different compounds and even a single strain can contain different desired compounds. Diatoms, like P. tricornutum , produce the carotenoid fucoxanthin, the omega‐3‐fatty acid eicosapentaenoic acid (EPA), and the 1,3‐β‐glucan chrysolaminarin (CRY) (Gao et al., 2017 ). Chrysolaminarin is a water‐soluble (1,3)‐(1,6)‐β‐ d ‐glucan, with antitumoral activity (Kusaikin et al., 2010 ). It promotes the health of juvenile fish (Reis et al., 2021 ). Furthermore, in recent studies with zebrafish, chrysolaminarin from P. tricornutum showed positive results against hypercholesterolemia, similar to the drug simvastatin, which is used to manage high cholesterol levels (Gora et al., 2022 ). Chrysolaminarin‐rich biomass also showed gut‐related benefits in a mouse study, such as an increase in short‐chain fatty acids (Stiefvatter, Neumann, et al., 2022 ). This makes it interesting for human nutrition as well. Further potential positive effects could already be shown in humans for example potential beneficial effects for healthy aging (Stiefvatter, Frick, et al., 2022 ). Chrysolaminarin closely resembles laminarin, a 1,3‐β‐glucan derived from macroalgae (Beattie et al., 1961 ). For laminarin, further possible applications have already been published, such as immunomodulatory properties or the promotion of animal health (Heim et al., 2015 ; Neyrinck et al., 2007 ; Sakai, 1999 ). If included in animal feed, it might contribute to the substitution of antibiotics in livestock farming (Lynch et al., 2010 ; Neyrinck et al., 2007 ). Laminarin also stimulates the response of vascular plants against pathogenic fungi and prevents fungal infections (Aziz et al., 2003 ; Cheong et al., 1991 ; Klarzynski et al., 2000 ). It can therefore be used in agriculture as well. Fucoxanthin is a xanthophyll that acts as a light‐harvesting pigment in the chloroplasts (Peng et al., 2011 ). Besides its antioxidative, anti‐inflammatory, and weight‐reducing properties, it shows activity against nonalcoholic fatty liver disease (NAFLD) (Fung et al., 2013 ; Gille et al., 2019 ; Heo et al., 2012 ; Hosokawa et al., 2004 ; Kotake‐Nara et al., 2001 ; Maeda et al., 2005 , 2006 ; Neumann, Louis, et al., 2018 ; Sachindra et al., 2007 ). The first product against NAFLD containing fucoxanthin is already available in the U.S. (Fucovital™; Algatech). EPA is an important omega‐3 fatty acid for human nutrition and is already used as a food supplement (Ritter et al., 2013 ). It shows antioxidative and anti‐inflammatory effects in humans and animals (Calder, 2010 ; Kim & Chung, 2007 ). It has been published that it has a positive effect on cardiovascular diseases and high blood pressure and might prevent the development of hypertension (Connor, 2000 ; Frenoux et al., 2001 ; Kang & Leaf, 1996 ; Narayan et al., 2006 ; Prisco et al., 1998 ). Chrysolaminarin serves as an energy and carbon storage compound in diatoms and is especially accumulated under nutrient‐depleted cultivation conditions, for example, nitrogen or phosphorous depletion (Frick et al., 2023 ; Gao et al., 2017 ; Kroth et al., 2008 ; Myklestad, 1989 ). Therefore, the production process for chrysolaminarin is composed of a nutrient‐replete phase (for biomass growth) and a nutrient‐depleted phase (usually nitrogen depletion) for chrysolaminarin accumulation. N‐depleted cultivation conditions are negative for the production of fucoxanthin and EPA, as it has already been reported that the fucoxanthin content as well as the EPA content are decreasing under N‐depleted conditions (Alipanah et al., 2015 ; Chrismadha & Borowitzka, 1994 ; Gao et al., 2017 ; Guo et al., 2016 ). Furthermore, the volumetric productivity of fucoxanthin and EPA is lower under N‐depleted conditions compared to nutrient‐replete conditions (Frick et al., 2023 ). However, even after a longer period of N‐depletion, EPA and fucoxanthin are still present in the biomass (Gao et al., 2017 ). Chrysolaminarin serves as the primary energy storage product of P. tricornutum (Alipanah et al., 2015 ; Granum & Myklestad, 2002 ; Myklestad, 1989 ). Additionally, for chrysolaminarin, P. tricornutum accumulates triglycerides, containing specifically C16 fatty acids (C16:0 and C16:1) during nutrient depletion, which serve as energy storage as well (Yodsuwan et al., 2017 ). Other than fatty acids, fucoxanthin, and EPA, chrysolaminarin was not considered in most studies regarding its production with microalgae (Yang et al., 2020 ). Only little is published on the production of chrysolaminarin in photobioreactors using P. tricornutum (Frick et al., 2023 ; Gao et al., 2017 ). Moreover, the influence of different cultivation conditions on chrysolaminarin accumulation in photobioreactors has not been investigated. In experiments using Skeletonema costatum grown in conical flasks (250 mL) Vårum and Myklestad already found an effect of different PFDs on the accumulation of chrysolaminarin (Vårum & Myklestad, 1984 ). Since chrysolaminarin is produced as energy storage, it can also be expected that the loss of energy from the increased self‐shading effects that occur at higher biomass concentrations has a (negative) effect on the accumulation of chrysolaminarin. However, it is not known (and not quantified) how I spec and biomass density affect the accumulation of chrysolaminarin in photobioreactors, especially in the nutrient‐depleted phase of a chrysolaminarin production process. Here we examined the influence of the light conditions on the accumulation of chrysolaminarin during nitrogen depletion (N‐depletion) in P. tricornutum cultures grown in commercially available, scalable flat‐panel airlift (FPA) reactors with artificial illumination. We focused on the influence of I \n spec and culture density. Besides chrysolaminarin, fatty acids were analyzed due to their role as energy and carbon storage in P. tricornutum . Fucoxanthin and EPA were analyzed as well, to examine the effects of the tested cultivation conditions on other potentially valuable products in the biomass. Although N‐depleted cultivation conditions are negative for the production of fucoxanthin and EPA, are both possible co‐products, which can be obtained from the produced biomass via cascaded extraction (Derwenskus, Weickert, et al., 2020 ; Gao et al., 2017 ). During the proposed phototrophic process, carbon dioxide would be fixed. Furthermore, because of the artificially illuminated closed reactor system, no surface area of (arable) land is required and the water consumption is low (Moomaw et al., 2017 ). Moreover, for the cultivation of the chosen organism ( P. tricornutum ), salt water can be used as the base of the cultivation media."
} | 3,001 |
21906329 | PMC3159908 | pmc | 6,025 | {
"abstract": "Genetically engineered Saccharomyces cerevisiae strains are able to ferment xylose present in lignocellulosic biomass. However, better xylose fermenting strains are required to reach complete xylose uptake in simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic hydrolyzates. In the current study, haploid Saccharomyces cerevisiae strains expressing a heterologous xylose pathway including either the native xylose reductase (XR) from P. stipiti s, a mutated variant of XR (mXR) with altered co-factor preference, a glucose/xylose facilitator (Gxf1) from Candida intermedia or both mXR and Gxf1 were assessed in SSCF of acid-pretreated non-detoxified wheat straw. The xylose conversion in SSCF was doubled with the S. cerevisiae strain expressing mXR compared to the isogenic strain expressing the native XR, converting 76% and 38%, respectively. The xylitol yield was less than half using mXR in comparison with the native variant. As a result of this, the ethanol yield increased from 0.33 to 0.39 g g -1 when the native XR was replaced by mXR. In contrast, the expression of Gxf1 only slightly increased the xylose uptake, and did not increase the ethanol production. The results suggest that ethanolic xylose fermentation under SSCF conditions is controlled primarily by the XR activity and to a much lesser extent by xylose transport.",
"introduction": "Introduction The yeast Saccharomyces cerevisiae has been extensively engineered for ethanolic fermentation of the pentose sugar xylose either by introducing genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), or by introducing the gene encoding xylose isomerase (XI) ( Hahn-Hägerdal et al. 2007 ; Van Vleet and Jeffries 2009 ; Matsushika et al. 2009 ). The aim is to achieve economically feasible ethanolic fermentation of hardwood and/or agricultural lignocellulose feedstock, since these raw materials have a high content of pentose sugars, primarily xylose (up to 20% of the dry matter) (USDE-database). Still xylose fermentation with recombinant S. cerevisiae is significantly less efficient than hexose fermentation. Among others this has been ascribed to the difference in cofactor preference of XR and XDH, which results in xylose to xylitol conversion rather than ethanolic fermentation ( Bruinenberg et al. 1983 ). Site directed mutagenesis has been applied on the XR to change the co-factor affinity, e.g. ( Watanabe et al. (2007) ). A different approach was used by ( Runquist et al. 2010a ) who arrived at a mutated version of the XR with changed kinetic properties using a random method in combination with a selection system. The mutant XR (N272D) from Pichia stipitis (mXR) has an increased ratio of NADH/NADPH utilization and an order of magnitude higher V max compared to the native enzyme. The introduction of mXR in S. cerevisiae otherwise engineered for xylose fermentation translated directly into increased ethanol yield and ethanol productivity and reduced xylitol formation in synthetic medium. Slow xylose fermentation has also been ascribed to be the less efficient xylose transport. In S. cerevisiae xylose and glucose compete for the same transport systems ( Kilian and Uden 1988 ; Meinander and Hahn-Hägerdal 1997 ) and the affinity for xylose is orders of magnitude lower than for glucose ( Kötter and Ciriacy 1993 ; Saloheimo et al. 2007 ; Gárdonyi et al. 2003 ). Several homologous and heterologous xylose transporters have been expressed in S. cerevisiae ( Hamacher et al. 2002 ; Saloheimo et al. 2007 ; Runquist et al. 2009 ; Katahira et al. 2008 ; Hector et al. 2008 ). Among the heterologous transporters the glucose/xylose facilitator Gxf1 from Candida intermedia ( Leandro et al. 2006 ) proved to have the highest transport capacity, which was reflected in the highest aerobic xylose growth rate ( Runquist et al. 2010b ). Gxf1 has also been expressed in the industrial xylose fermenting S. cerevisiae strain TMB3400 (Fonseca et al. submitted). Its presence increased xylose consumption in simultaneous saccharification and co-fermentation (SSCF) of acid-pretreated wheat straw, however, without increasing the ethanol yield. Simultaneous saccharification and fermentation (SSF) ( Takagi et al. 1977 ) has been established as a promising option for ethanol production from lignocellulosic materials ( Olofsson et al. 2008a ) since the overall ethanol yield has been reported to be higher than if the enzymatic hydrolysis and fermentation are carried out separately (SHF) ( Wingren et al. 2003 ). Furthermore it also been established that xylose consumption increases in SSF ( Öhgren et al. 2006 ; Olofsson et al. 2008b ), which has therefore been re-named SSCF also to include co-fermentation of hexose and pentose sugar. The current study was undertaken to investigate to what extent the presence of mXR instead of XR would allow ethanolic fermentation of the additional xylose taken up by strains carrying the Gxf1 facilitator (Fonseca et al. submitted). Therefore, isogenic haploid S. cerevisiae CEN.PK strains expressing a heterologous XR/XDH/XK pathway were constructed. In addition to the control strain carrying the native XR, strains carrying mXR, Gxf1 and both mXR and Gxf1 were generated. The strains were assessed in SSCF of acid-pretreated wheat straw. The presence of mXR significantly increased xylose uptake and ethanolic xylose fermentation and reduced xylitol formation. In contrast, Gxf1 either alone or together with mXR, at most increased xylose uptake with about 10% leaving the ethanol formation unchanged.",
"discussion": "Discussion The glucose/xylose facilitator Gxf1 from C. intermedia ( Leandro et al. 2008 ) has been shown to increase xylose uptake and aerobic growth at low sugar concentrations in an laboratory xylose-utilizing CEN.PK strain ( Runquist et al. 2009 ) as well as in the industrial xylose-utilizing TMB3400 strain (Fonseca et al. submitted). Similarly, the presence of the mutated (N272D) xylose reductase (mXR) from P. stipitis , increased xylose uptake and anaerobic growth ( Runquist et al. 2010a ) in synthetic medium. In addition, mXR shifted product formation from xylitol to ethanol. The current comparison using isogenic S. cerevisiae CEN.PK strains was undertaken to elucidate the relative contribution of these two beneficial genetic modifications on xylose consumption and ethanol and clarify if these genetic traits could act synergistically. Simultaneous saccharification and co-fermentation (SSCF) ( Olofsson et al. 2008a ) of non-detoxified pretreated wheat straw was chosen as experimental model, since it is an industrial medium, interesting for commercial ethanol production scale. Our investigation showed that in the CEN.PK strain background and in the SSCF set-up, mXR had a far greater influence on xylose consumption and product formation than Gxf1. The presence of mXR doubled the xylose uptake, decreased the xylitol yield by half and as a result increased the obtained ethanol yield in SSCF by about 20%. In contrast, Gxf1 at most increased the xylose uptake by 10% irrespective of the presence of XR and mXR, receptively. SSF (simultaneous saccharification and fermentation), the forerunner of SSCF was originally designed as a means to generate low glucose concentration in the reactor to overcome glucose inhibition of cellulose hydrolysis ( Takagi et al. 1977 ). It was later observed that this set-up also favored co-utilization of xylose when recombinant xylose-utilizing strains of S. cerevisiae were used ( Olofsson et al. 2008b ; Öhgren et al. 2006 ). In SSCF, the fermenting yeast is exposed to a high xylose/glucose ratio since the hemicellulose fraction is primarily hydrolyzed in the acid-pretreatment step ( Olofsson et al. 2008a ) while glucose is continuously released throughout the enzymatic hydrolysis. Enhanced co-utilization of xylose and glucose in SSCF is in accordance with numerous independent observations, which demonstrated that glucose in fact enhances xylose utilization at low but non-zero concentrations ( Meinander et al. 1999 ; Pitkänen et al. 2003 ; Krahulec et al. 2010 ). This has been attributed both to activation of the enzymes of the lower glycolytic pathway ( Boles et al. 1996 ), and to improved co-factor regeneration ( Pitkänen et al. 2003 ). In addition the low glucose concentration in SSCF favors induction of high affinity hexose transporters, which also display high affinity for xylose ( Pitkänen et al. 2003 ; Bertilsson et al. 2008 ). Therefore the fact that xylose uptake only increased by 10% in the Gxf1 strains may not only reflect the properties of the transporter, but may also result from the SSCF conditions. When the Gxf1 transporter was expressed in the industrial S. cerevisiae strain TMB3400 and assessed in SSCF of acid-pretreated wheat straw similar to the current experimental set-up, the xylose uptake also increased by about 10% (Fonseca et al. submitted). The additional xylose taken up was stoichiometrically converted to xylitol and glycerol. Metabolic flux analysis (MFA) suggested that the presence of Gxf1 shifted the control of xylose catabolism from transport to down-stream catabolic reactions. The mXR mutant has a higher V max and higher NADH/NAPH selectivity ratio, which was shown to directly relate to increased anaerobic xylose growth and increased ethanol formation ( Bengtsson et al. 2009 ; Runquist et al. 2010a ). The current study was set up to investigate whether the presence of mXR would shift the control of xylose catabolism to transport. However, the results show that xylose catabolism downstream of transport still dictated the metabolic flux, and that an even faster xylose catabolism would be required to fully benefit from the increased xylose transport capacity. The presence of only Gxf1 resulted in slightly higher xylose consumption, which was not converted to ethanol. Instead somewhat less ethanol was produced, which was not seen when mXR was also expressed. This may reflect that transport exercises a slightly higher control in the strain harboring mXR because mXR has significantly higher activity than XR ( Runquist et al. 2010a ) which is in accordance with previous reports showing that transport becomes more controlling at higher XR activity ( Gárdonyi et al. 2003 ). During pretreatment and hydrolysis a spectrum of compounds that inhibit the cellular metabolism are released and formed and many of these compounds inhibit ethanolic fermentation ( Almeida et al. 2007 ). S. cerevisiae strains with an industrial background are generally more inhibitor tolerant than haploid laboratory strains ( Almeida et al. 2007 ). The haploid CEN.PK strain background was chosen in the current study to generate isogenic strains that permitted the assessment of the relative contribution of mXR and Gxf1, respectively, to ethanolic xylose fermentation in SSCF of pretreated wheat straw. The control strain carrying the native XR consumed 38% of the available xylose, whereas the mXR strain converted twice as much in the non-detoxified wheat straw. The conversion obtained with the mXR strain in fact compared well to that reported for the industrial XR/XDH based xylose fermenting strain TMB3400 in SSCF of pretreated wheat straw of a similar composition ( Olofsson et al. 2008b ). Among the inhibitory compounds formed during pretreatment and hydrolysis, there are several which act as electron acceptors ( Almeida et al. 2007 ). Such compounds have been shown to function as \"redox sinks\" able to alleviate the redox imbalance caused by the difference in cofactor preference of XR and XDH ( Wahlbom and Hahn-Hägerdal 2002 ). This has been shown to reduce the xylitol yield in non-detoxified hydrolyzate with as much as three times in model SSF experiments compared to defined media ( Olofsson et al. 2008b ). For the mXR strain xylitol formation was reduced about 50% from 0.24 to 0.13 g g -1 when compared with xylose fermentation in defined medium ( Runquist et al. 2010a ). In conclusion, the current work investigated targeted metabolic changes for improved xylose fermentation in SSCF of undetoxified pretreated wheat straw. These kinds of investigations are important since strain-improvements are often considerably less pronounced in lignocellulosic hydrolyzates under process-like conditions. Due to the mutated XR the xylose uptake could be doubled along with a significant reduced xylitol yield, resulting in a substantial increase in the ethanol yield. It will be important to increase the final ethanol concentration further by increasing the WIS-content with a maintained ethanol yield for the economic viability of the process ( Galbe et al. 2007 ). This is likely to require a combination of further strain development and improved process technology."
} | 3,200 |
28676652 | PMC5496868 | pmc | 6,027 | {
"abstract": "The deep biosphere is one of the least understood ecosystems on Earth. Although most microbiological studies in this system have focused on prokaryotes and neglected microeukaryotes, recent discoveries have revealed existence of fossil and active fungi in marine sediments and sub-seafloor basalts, with proposed importance for the subsurface energy cycle. However, studies of fungi in deep continental crystalline rocks are surprisingly few. Consequently, the characteristics and processes of fungi and fungus-prokaryote interactions in this vast environment remain enigmatic. Here we report the first findings of partly organically preserved and partly mineralized fungi at great depth in fractured crystalline rock (−740 m). Based on environmental parameters and mineralogy the fungi are interpreted as anaerobic. Synchrotron-based techniques and stable isotope microanalysis confirm a coupling between the fungi and sulfate reducing bacteria. The cryptoendolithic fungi have significantly weathered neighboring zeolite crystals and thus have implications for storage of toxic wastes using zeolite barriers.",
"introduction": "Introduction The deep subsurface biosphere comprises microorganisms several kilometers below the surface 1 . Microbiological investigations have revealed active deep ecosystems in marine sediments 2 , deep-sea hydrothermal vents 3 , sub-seafloor igneous rocks 4 , and terrestrial sedimentary 5 and crystalline rocks 6 . Owing to its recent discovery and the difficulties in accessing samples, the deep biosphere is among the least understood ecosystems on Earth. Although processes are relatively slow because of the low energy supply 7 the deep ecosystems are proposed to comprise a significant biomass and play an important role in the energy cycling of the Earth 8 . Estimates from the deep continental subsurface suggest that this environment accommodates a significant part of the Earth’s biomass (up to 19%) 8 . Until recently, the majority of the microbiological investigations in the deep biosphere have been focused on prokaryotes, and the potential presence of eukaryotes such as fungi has been largely neglected 9 , 10 . Recently, fungi have been found to exist and have been isolated from various deep marine settings 10 – 14 including sub-seafloor basalt 9 , 15 – 17 , suggesting that fungi play a major role in the element and energy cycling 9 . In continental deep settings studies involving fungi are surprisingly rare. Reitner et al. 18 described fossilized putative fungal hyphae in the Triberg granite, Germany, and Ivarsson et al. 19 described fossilized mycelia from the Lockne impact structure, Sweden, of Ordovician age. Ekendahl et al. 20 isolated a few strains of yeast fungi from fluids at Äspö, Sweden, and Sohlberg et al. 21 examined the total fungal diversity in anaerobic bedrock fractures at Olkiluoto, Finland, and found the diversity to be higher than expected consisting of most major fungal phyla, some minor phyla and even novel species. At great depth in continental granite aquifer systems anoxic conditions prevail, and the fungi living there are considered anaerobic 21 . Anaerobic fungi are so far poorly understood in an environmental context, reported only from a few anoxic settings 14 , 22 , but are most thoroughly described from rumina of ruminating herbivores 23 , 24 . Because of the production of H 2 during their respiration, anaerobic fungi consort with H 2 -dependent methanogenic and acetogenic archaea in the rumen, which enhances growth of both organisms. Potentially any H 2 -dependent chemoautotrophic microorganism could be fuelled by anaerobic fungi in an anoxic environment 25 and it has been suggested that anaerobic fungi represent a neglected geobiological force in the subsurface ecosystem 9 . Direct evidence of such consortia in the subsurface remains to be confirmed. However, the fungi that form symbiotic relationships with acetogens and methanogens in the rumen have recently been described from marine sediments 26 . Even though the presence of fungi in the continental crystalline basement is confirmed by a few reports, there is a huge gap in knowledge compared to what is known about the prokaryotes, and there is an urgent need to investigate the abundance, diversity and ecological role of fungi in these deep environments. Here we present an extensive study of previously unseen organically preserved and partly mineralized fungal hyphae, inferred as anaerobic fungi, from deep fractured continental crystalline rocks (740 m depth at the Laxemar site in Sweden). Utilization of state-of-the-art methodology including secondary ion mass spectrometry (SIMS), synchrotron radiation X-ray tomographic microscopy (SRXTM), and Time-of-Flight (ToF-) SIMS enables comprehensive new characterisation of the fungi, the cryptoendolithic behaviour of fungi, and the prokaryote-fungus interaction in this environment. This not only increases the understanding of the role fungi play in the energy cycling of the deep biosphere, but also has societal implication for long-term storage of toxic wastes.",
"discussion": "Discussion The mycelium-like appearance, the diameter, and the anastomosing behavior of the filaments (Fig. 1 ) are all distinctive features of fungi. The presence of a mineralized central strand has been shown as a common feature among fossilized fungal hyphae 33 – 36 . Lining of mineral surfaces by a basal biofilm from which further hyphal growth emanates to form a mycelium is also typical for endolithic fungi 15 , 19 , 33 , 34 . Except for fungi, actinobacteria and the stramenopile oomycetes are the only microorganisms forming mycelium-like networks of branching filaments. However, actinobacterial filaments never exceed 2 µm in diameter and anastomoses have not been confirmed 37 , 38 . Thus, the diameter of the current filaments, together with the presence of anastomoses excludes an actinobacterial interpretation. Oomycete filaments have been reported to form anastomoses, but as a means of conjugation rather than as a structural feature 39 . Based on these morphological features we infer a fungal interpretation of the networks and suggest that they represent diagenetically mineralized fungal hyphae. As further support for this, the weathered mineral surfaces in contact with the fungi bear close resemblance to fungal induced weathering seen in many other minerals, owing to production of organic acids 40 . Active boring seen among fungi in sub-seafloor basalts is not observed in the sample of the present study. However, the fungal hyphae typically exploit microfractures in the minerals for penetration. The partly mineralized nature is in itself a rare finding, which has previously only been observed in the laboratory 41 , 42 . Usually, subsurface fungi are completely fossilized to clay minerals and Fe-oxides with rare carbonaceous elements and no biomarkers at all 33 , 34 , except rare chitin observations 16 . Our findings give insights into the fossilization process of fungi through a transition from maturation of the organic matter to a carbonaceous material, before being finally mineralized by clays and Fe-oxides. Based on our observations the mineralization starts from the centre of the hypha with a fully mineralized Fe-oxide dominated central strand and clay-mineral dominated margins. The negatively charged carbonaceous material attracts the positively charged Si, Al, Mg, Fe (and minor Na, and Ca) cations of the clays. Initial adsorption of cations on the carbonaceous hyphae sparks subsequent adsorption and clay mineralization 34 , 43 . The end result of complete mineralization by clays and Fe-oxides is in agreement with previous observations of deep fossilized fungi and supports what seems to be an overall characteristic pattern of fungal fossilization in deep ecosystems. Fungi are heterotrophs and need access to carbohydrates like mono- or polysaccharides for their metabolism. In the deep oligotrophic granite environment, the most likely source of carbohydrates is living or dead bacterial biofilms 44 . The large 13 C-depletions of the calcite, resulting in δ 13 C values as low as −43‰ (Fig. 7c ), point to oxidation of methane in the fracture system 45 , as described previously for this setting 30 . The youngest generation of calcite shows δ 13 C values suggesting microbial degradation of organic C. Remnant biofilms of metanotrophs, as well as sulfate-reducing bacteria (SRB) that evidently have occupied the fracture at several occasions, may have acted as nutrients for the fungi and triggered the fungal colonization of the system. The fungi cannot be taxonomically classified in detail because of the fossilization and lack of morphological features like septa, but they are considered to have grown in an anaerobic environment. A major support for this is that the current groundwater in the fracture network turns anoxic in the upper tens of meters 46 and below that depth remains strictly reducing, with Eh values in the range of −300 to −200 mV 47 . Based on these features, it seems highly unlikely that the conditions in the paleo-groundwater would have been oxidising or suboxic at a depth as great as 740 m. There are several additional lines of support for prolonged anoxic conditions. First, there is pyrite in relation to the hyphae; second, oxidation-related alteration features are not detected in any of the pyrite generations in the fractures, in contrast to pyrite at shallower depth where waters with dissolved oxygen have infiltrated 46 ; third, Ce(IV) and positive Ce anomalies, which are indicative of oxidising conditions, are frequent in fracture coatings in the upper 10 m of the bedrock but absent below that depth 48 ; and, fourth, abundant signs of anaerobic oxidation of methane ( 13 C-depleted calcite, in this fracture and elsewhere in the fracture network 30 ). There is thus strong evidence that the fungi were anaerobic, in a manner similar to fungi filtered from deep-water samples from fractured crystalline rocks in Finland 21 . The partly mineralized nature of the fungi and the degraded and matured carbon in the fungi speak against a modern origin. The only timing indication available is offered by the fluid inclusions in the calcite showing <50 °C, which rule out formation prior to the Mesozoic era based on the uplift history of the area 49 , 50 , but it should be emphasized that this is a maximum age estimate and not a direct age determination. In addition, the calcite crystals show fluid inclusion salinities that are much higher than in the present groundwater. The S − -isotope signatures indicate that the older pyrite generation precipitated from a more homogeneous fluid than the fine-grained younger generation of pyrite (Fig. 6 ). The relatively small variation in δ 34 S of the older pyrite generation likely reflects formation during bacterial sulfate reduction (BSR) at relatively open system conditions. The younger fine-grained pyrite has a more clear relation to the hyphae than the older pyrite, and, hence, the discussion about the hyphae-SRB relation only takes the younger of these two pyrite generations into account. These fine-grained pyrite crystals were produced via the activity of SRB, because the low δ 34 S values,are diagnostic for microbial transformation of sulfate to sulfide as 32 S SO4 is favoured over 34 S SO4 in the SRB metabolism 51 . The large span in δ 34 S of this pyrite generation is interpreted as the result of Rayleigh type distillation where the δ 34 S composition of the sulfate and thus of the precipitated pyrite became progressively higher as the sulfate pool was exhausted during BSR under closed system conditions 51 . Coexistence of the SRB producing these pyrite crystals and the fungi is thus possible and supported by a number of features. First, pyrite grows on original hyphal walls and not on parts that have been exposed by later breakdown/degradation caused by the core drilling or sample preparation, and clusters of pyrite enfold hyphae, forming almost a girdle-like structure that follows the hyphal morphology tightly (Fig. 5b, c ); second, lack of hyphal degradation at the direct contact with the pyrite and no sign of pervasive replacement of hypha by pyrite, which is the common case in complete pyritization of fossils caused by heterotrophic bacterial activity 52 , 53 , argue against a situation where SRB only scavenged the fungal biomass; third, hyphal growth has been influenced by the presence of pyrite crystals; for example, hyphae have grown around existing pyrite that sits upon other hyphae (Fig. 5c ) and pyrite crystals are partly enclosed by the hyphae (Fig. 5d, e ); and fourth, spatial relation between pyrite, fatty acids and sugar compounds that resemble chitin, as revealed by Tof-SIMS analyses. Anaerobic fungal species have no mitochondria and are unable to produce energy by either aerobic or anaerobic respiration 54 , 55 . Instead, anaerobic fungi have hydrogenosomes, and produce mainly H 2 , but also formate, lactate, acetate and carbon dioxide, as metabolic waste products 54 , 56 . Anaerobic fungi consort with H 2 -dependent methanogenic archaea in the rumen of ruminants, but potentially any H 2 -dependent chemoautotrophic microorganism could be fuelled by anaerobic fungi in an anoxic environment 25 , for instance SRB. Although the largest S-isotope fractionations observed in pure culture experiments have been associated with heterotrophic BSR 57 , autotrophic BSR using H 2 also involves significant S-isotope fractionation (δ 34 S H2S -δ 34 S SO4 of up to 37‰), particularly at low H 2 concentrations and slow BSR rates 58 . Because the in situ rate of bacterial processes and generally also the concentrations of H 2 appear to be substantially lower in the granitic fractures 6 , 59 than those manipulated in the laboratory, larger fractionation than reported from the laboratory appears reasonable under the extreme oligotrophy in the deep granite fractures. Hence, H 2 is a plausible electron donor for the SRB that produced the younger generation of pyrite, in line with the fact that the current groundwater at the site carries autotrophic microorganisms alongside heterotrophic ones 27 . We accordingly propose that H 2 , and potentially some other substrate such as acetate, provided by anaerobic fungi, have triggered SRB growth (Fig. 8 ) and that, consequently, the intimate relationship between the fungal mycelium and the pyrite crystals represents a fossilized consortium of anaerobic fungi and SRB being the first record of these previously hypothesized communities 25 . This further suggests that the deep oligotrophic biosphere in crystalline continental rocks may be a neglected vast fungal habitat. Fig. 8 Conceptual model of coexistence and growth of fungi and sulfur reducing bacteria deep in crystalline bedrock. Growth of hypha starts at the biofilm on the zeolite surface, where sulfate reduction by sulfate-reducing bacteria (SRB) and anaerobic oxidation of methane (AOM) has led to oversaturation and precipitation of older calcite and pyrite. The biofilm provides organic carbon (C x H 2 y O y ) for the fungi to build biomass. The growing hypha metabolizes dissolved organic carbon both from the water and from the syntrophic SRB, which in turn thrive from the H 2 produced by the fungi. The SRB reduce sulfate (SO 4 \n 2− ) dissolved in the water autotrophically and produce HS − which reacts with dissolved Fe 2+ to form pyrite (now present on the hypha). The SRB use CO 2 from both the fungal metabolism and dissolved CO 2 in the water as carbon source \n Hydrogen gas has been proposed to be an important substrate for deep subsurface lithoautotrophic ecosystems 60 – 63 and a limiting factor for the persistence of an indigenous SRB community 64 , 65 . However, the formation and origin of H 2 remain elusive, and several different processes have been proposed, including radiolysis 60 over long time spans in the subsurface environment. Investigations from the Fennoscandian shield show highly variable H 2 concentrations in the deep groundwater (down to 1000 m depth) with concentrations up to 190 µl/l 27 , 59 , that are correlated with neither depth nor residence times of the waters, which in several cases are in the order of just a couple of thousand years 66 . Theseare certainly too short time periods for build-up of significant H 2 concentrations by radiolysis (cf. ref. 60 ) implying that radiolysis is not the sole source of the elevated H 2 concentrations. Instead, based on our findings and ambiguous traces of fungi in the deep aquifer at Olkiluoto, Finland 21 , we propose that subsurface fungi are neglected and likely significant providers of H 2 for autotrophic microbial processes in the oligotrophic crystalline continental crust. Anaerobic fungi could potentially pose an environmental threat to barriers in geological repositories of toxic wastes, via two mechanisms: mediating direct bioerosion of the barrier system by chemical dissolution, as well as supporting an H 2 -dependent SRB community capable of causing corrosion to copper canisters containing spent nuclear fuel 67 . Regarding the first mechanism, the extensive weathering of zeolites seen in the granite fracture in the present study, as well as similar observations in sub-seafloor basalts elsewhere 33 , 34 , calls for consideration when planning to use this group of minerals as geochemical barriers in subsurface storages. Zeolites have been planned to function as an ion-exchange retention barrier for the storage of high-level nuclear waste in the US 68 , 69 . The cryptoendolithic behavior of anaerobic fungi may challenge the long-term stability of such systems, at least at low-temperatures. Regarding the second mechanism, our study shows a previously unseen relation between fungi and SRB at great depth in fractured granite which may, alongside long-term radiolytic H 2 consumption coupled to sulfate reduction, enhance sulfide levels in the deep groundwater aquifer. The ultimate result may be higher rates of sulfide-induced copper corrosion and CuS formation 64 , 65 . The recognition of fungi in the subsurface realm, thus, indicates the presence of a previously neglected geobiological agent, the environmental impact and societal implications of which have yet to be accounted for."
} | 4,594 |
27876549 | PMC5221347 | pmc | 6,028 | {
"abstract": "The family Leguminosae comprises approximately 20,000 species that mostly form symbioses with arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria (NFB). This study is aimed at investigating and confirming the dependence on nodulation and biological nitrogen fixation in the specie Piptadenia gonoacantha (Mart.) Macbr., which belongs to the Piptadenia group. Two consecutive experiments were performed in a greenhouse. The experiments were fully randomized with six replicates and a factorial scheme. For the treatments, the two AMF species and three NFB strains were combined to nodulate P. gonoacantha in addition to the control treatments. The results indicate this species’ capacity for nodulation without the AMF; however, the AMF + NFB combinations yielded a considerable gain in P. gonoacantha shoot weight compared with the treatments that only included inoculating with bacteria or AMF. The results also confirm that the treatment effects among the AMF + NFB combinations produced different shoot dry weight/root dry weight ratios. We conclude that AMF is not necessary for nodulation and that this dependence improves species development because plant growth increases upon co-inoculation.",
"conclusion": "Conclusions This article concluded that mycorrhizal fungi are not necessary for nodulation in the species P. gonoacantha , which highlights the effect of a substrate on forming any symbiotic association because the substrate used herein was different. The co-inoculated P. gonoacantha plants produced a higher shoot weight with D. heterogama and BSP1 strain. This combination was very promising for P. gonoacantha inoculation in the future for seedlings. These results stress the relevance of studies of inoculation in tree species used in reforestation and land reclamation.",
"introduction": "Introduction The family Leguminosae comprises approximately 3000 species throughout Brazil and is the third largest angiosperm family, with approximately 20,000 species and 700 genera, 1 only surpassed by Orchidaceae and Asteraceae . 2 Most species are associated with nitrogen-fixing microorganisms and arbuscular mycorrhizal fungi (AMF), 3 , 4 which are the two main symbiotic microorganisms in terrestrial plants. New microbial species and rhizobia plant infection mechanisms were discovered through studying this bacterial diversity. 5 , 6 Certain native legume species in the subfamily Mimosoideae exhibit atypical characteristics with a high exploitation potential for the two symbioses. Synergy between the symbionts has also been reported; mycorrhizal fungi can aid in increasing biological nitrogen fixation, and nitrogen-fixing bacteria influence mycorrhizal colonization. 7 , 8 The species studied herein, Piptadenia gonoacantha (Mart.) J. F. Macbr., is arboreal and naturally occurs in southern and southeastern Brazil. It is economically and socially useful because it is used in the furniture construction, energy, cellulose, and paper sectors, among other fields. 10 This species is also used considerably in degraded site restoration projects because it can biologically fix nitrogen. 11 A recent discovery showed that the legumes P. gonoacantha and Piptadenia paniculata , both Atlantic Rainforest natives, 12 did not nodulate in pots with soil and sand as substrates when they were not co-inoculated with AMF. 11 Asai 13 reported this observation and indicated that certain legumes do not nodulate in autoclaved soils without co-inoculation by mycorrhizal fungi. Crush 14 provided the first conclusive observations on this synergistic effect. However, the effects of the substrates on symbiosis formation remains uncertain; most likely, an underabundance of phosphorus limits symbiosis formation even if mycorrhizae are necessary for nodulation because phosphorus is important for forming nodules in the root system. In addition to affecting nodulation, the mycorrhizal fungus aids in better development of the plant species because biological nitrogen fixation demands high levels of energy, which is provided by the plant as ATP. However, the great phosphorus deficiency in tropical soils limits the maximum development of the symbiosis. Thus, increased phosphorus absorption by AMF yields increased fixation. 2 , 8 The synergistic effect between the symbionts is evident from the phosphorus concentration in the nodules, which is up to three times higher than in other organs. 15 This link is also attributed to the number of genes and root exudates that the symbioses share. This evidence supports the hypothesis that the symbioses formed with legume family species were inherited from mycorrhizal fungi because two forms of symbiosis emerged at different evolutionary times during colonization by terrestrial plants. 16 From a functional perspective, bacterial and AMF compatibility can also alter symbiotic efficiency because the combination of inoculating with AMF and bacterial strains can either reduce or increase efficiency in certain bacterial strains. 17 , 8 , 9 This study is based on the hypothesis that the species P. gonoacantha depends on the mycorrhizal fungus for nodule formation, hypothesis described by the author Jesus et al. 11 Thereby, the article aimed at investigate and confirm the dependence of the specie on arbuscular mycorrhizal fungi for nodulation and biological nitrogen fixation.",
"discussion": "Discussion Jesus et al. 11 indicated that P. gonoacantha is highly dependent on AMF, especially for nodulation. However, the present study suggests otherwise because plants from this species nodulated without the mycorrhizal fungus, which was also observed by Bornaud et al. 8 Most likely, the different substrates in the studies affected the results; however, the phosphorus source may have been more crucial to the results than the substrate. Jesus et al. 11 used a small quantity of a slightly soluble source, rock phosphate; however, a nutrient solution with readily available phosphorus was used herein. Analyzed together, the results of these two experiments indicate that “pau-jacaré”( P. gonoacantha (Mart.) J. F. Macbr) greatly depends on mycorrhizae for growth and requires AMF when phosphorus is a limiting factor. However, when phosphorus is readily available, the plant no longer depends on the fungus to grow and can nodulate without it. However, this does not mean that co-inoculation is not important because visual observations of the nodules from plants not inoculated with AMF indicate that these nodules are amorphous. This amorphous characteristic indicates that the fungus leads to morphological changes and altered efficiency in these nodules, as demonstrated by the acetylene reduction assay in plants with and without the mycorrhizal fungus. Further, this characteristic is evident in the scientific literature, which indicates a positive interaction between the fungus and bacteria; bacteria favor colonization of P. gonoacantha roots by AMF, 8 and the fungus favors bacterial efficiency. 29 The different results for the different bacteria-AMF combinations indicate that certain combinations are more efficient than others, which suggests a certain specificity between the symbiotic microorganisms. These data corroborate Bournaud, 8 who observed that the mycorrhizal colonization rate of P. gonoacantha roots varied due to a co-inoculated rhizobial strain. The colonization rate in the different experiments may reflect strategic mycorrhizal colonization because D. heterogama should promote higher colonization of the root system compared with the fungal species with larger diameter spores relative to the duration of each experiment ( G. margarita ). The short duration of the experiments may have produced the different results for the different strategies. Fungal species under the K strategy establish in the root system over a longer time period due to the longer duration required for germination and development as well as the low number of spores produced, which was observed for G. margarita (W.N. Becker & I.R. Hall), exhibiting the lowest colonization rates. According to the efficiency and efficacy results in the second experiment, the D. heterogama and BSP1 strain combination promoted an approximately 10× higher gain in shoot weight, which demonstrates a high recommendation potential. 30 These results corroborate results from Bournaud et al., 8 who observed that the BSP1 strain provides the highest shoot growth, nodulation, and efficiency in co-inoculated P. gonoacantha plants. However, the author used Rhizophagus clarus (T.H. Nicolson & N.C. Schenck) C. Walker & A. Schübler. The plants inoculated with the BSP1 strain exhibited slightly higher root mycorrhization than plants inoculated with the same fungus plus the BR 4802 strain. These data also corroborate the results from Bournaud et al., 8 who observed that the BSP1 strain encourages root colonization by AMF and consequently increases nodule efficiency. Here, the effect observed for R. clarus was observed for D. heterogama . However, the accumulated dry weight and mycorrhizal colonization values were lower in plants inoculated with G. margarita , which indicates that the different fungus-bacteria combinations may not necessarily exhibit a positive effect on P. gonoacantha plant development. Co-inoculation does not ensure better development as observed herein. For example, Patreze et al. 31 and Carneiro et al. 3 observed low colonization for the species Anadenanthera colubrina (0–14%), A. falcata (20–49%), and A. peregrina (1–19%). In addition to nutritional factors, the variable infection rate results may be associated with specificity between the host plants and AMF. An analysis of the nitrogen controls (NC and AMF + NC) indicates the importance of the mycorrhizal fungus for better absorption of the added nitrogen. These results were confirmed during the second experiment, where the gain in shoot dry weight of the plants inoculated with G. margarita (W.N. Becker & I.R. Hall) was twice the weight gain as in plants that only received N. These observations highlight the mycorrhizal dependence of the P. gonoacantha species, not only for phosphorus absorption but also for other nutrients. 32 , 33 Three groups of means formed upon analyzing the SDW/ROOT ratio in the species P. gonoacantha ; the BSP1 strain without co-inoculation exhibited inferior behavior compared with the strain that contacts the two AMF species in the experiment. This contact altered the ratio in favor of greater shoot development. The analysis allows us to discern a range of possible responses between the microorganisms and their respective combinations."
} | 2,668 |
28345040 | PMC5345924 | pmc | 6,031 | {
"abstract": "A polymer is described that is conductive and stretchable, which can lead to electronics that can conform to the human body.",
"introduction": "INTRODUCTION Recent advancements in stretchable electronics have blurred the interfaces between human and machine ( 1 , 2 ). Devices such as epidermal electronics, implantable sensors, and hemispherical eye cameras all rely on the intimate contact between devices and curvilinear surfaces of various biological systems while operating with stability under up to 100% strain ( 3 – 5 ). The most successful concept leading to such devices builds on linking rigid islands of active components [that is, transistors, light-emitting diodes (LEDs), or photovoltaics] with stretchable interconnects ( 1 , 3 , 6 , 7 ). Hence, developing conductors that can retain good electrical performance under high mechanical strain is paramount. Stretchable conductors have been fabricated via two main routes: strain engineering and nanocomposites ( 1 , 8 , 9 ). In the first approach, nonstretchable inorganic materials, such as metals, are geometrically patterned into wavy lines that can be extended when the underneath elastomer substrate is stretched ( 10 ). Alternatively, depositing a thin layer of conducting materials such as metals, carbon nanotubes, or graphene on a prestrained substrate leads to the formation of periodic buckles upon the release of the strain, which allows the material to accommodate further cycles of stretching up to the initial prestrained value ( 5 ). A kirigami design or microcracks have also been applied to sheets of flexible materials to enable macroscopic stretching motion ( 11 – 13 ). These methods demonstrate the possibility of transforming virtually any rigid materials into stretchable materials while maintaining their electrical properties. However, the fabrication methods involved are usually complicated, and it is challenging to achieve high device density owing to the large geometric patterns required for high stretchability. In addition, the buckling and kirigami methods lead to out-of-plane patterns, which could be difficult to encapsulate and disadvantageous for devices that require planar interfaces or lower profiles. Embedding a conductive filler in an insulating elastomeric matrix to form a nanocomposite is the second major route toward stretchable conductors ( 8 , 14 ). Typically, one-dimensional materials such as carbon nanotubes (CNTs) and silver nanowires are chosen as the conductive fillers because of their high aspect ratios ( 15 – 17 ). Metal nanoparticles or flakes have also been shown to be good filler materials under specific conditions because of their ability to self-organize upon stretching ( 7 , 18 ). Despite the versatility and large number of material choices, the percolation-dependent conductivity is highly strain-sensitive and remains a hurdle for device miniaturization and cycling stability ( 8 ). To achieve a highly stretchable and highly conductive material that is readily solution-processable and patternable, an intrinsically stretchable conductor is desirable. Conducting polymers are good candidates because of the flexibility in tuning the molecular structures and electrical and mechanical properties. Their solution processability offers additional advantages for large-scale production of flexible electronics. Unfortunately, high conductivity and high stretchability have not been achieved simultaneously for conducting polymers ( 19 – 21 ). In general, to achieve high conductivity, high crystallinity and low insulating content are required. However, to render a polymer film stretchable, a high degree of disorder with chain folding is advantageous to create a large free volume for polymer chain movement and unfolding when being stretched ( 22 , 23 ). Poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) has the highest reported conductivity among solution-processed polymers but has a fracture strain of as low as ~5% ( 20 ). Previous efforts enabled the stretching of PEDOT:PSS by incorporating plasticizers such as Zonyl or Triton ( 19 – 21 ). However, the enhanced stretchability often results in much lower conductivities, and the value further decreases with the application of strain. As yet, the best reported value is 550 S/cm under 0% strain and a conductivity of 13 S/cm under a fracture strain of 188% ( 20 ). Hence, these materials have generally been used as pressure or strain sensors where a large change in electrical signal upon strain is desirable, but they fail to serve as interconnects. Interconnects for various rigid electrical components in circuits require an intrinsically stretchable conducting polymer with conductivity >1000 S/cm at >100% strain and minimal temperature dependence. Here, we demonstrate a method for creating highly stretchable and conductive PEDOT films with high cycling stability by incorporating ionic additives–assisted stretchability and electrical conductivity (STEC) enhancers ( Fig. 1 , A and B). The resulting PEDOT:PSS films are both highly conductive and stretchable (higher than 4100 S/cm under 100% strain), giving rise to transistor arrays up to five times higher in island-to-interconnect ratio as compared to those using wavy metal interconnects. Fig. 1 Chemical structures and schematic representation. ( A and B ) Chemical structures of PEDOT:PSS (A) and representative STEC enhancers (B) (see complete list in the Supplementary Materials). ( C and D ) Schematic diagram representing the morphology of a typical PEDOT:PSS film (C) versus that of a stretchable PEDOT film with STEC enhancers (D). ( E ) Photograph showing a freestanding PEDOT/STEC film being stretched. ( F and G ) Stress/strain (F) and strain cycling behavior (G) of freestanding PEDOT/STEC films.",
"discussion": "DISCUSSION We have demonstrated a highly stretchable and conductive PEDOT polymer by incorporating ionic additive–assisted STEC enhancers that result in a morphology that is beneficial for both high stretchability and conductivity. A further boost in conductivity is achieved through a proper selection of anions. The resulting materials show record-high stretchability and conductivity, the coexistence of which is rare for conducting polymers. High-density FET arrays connected using the stretchable PEDOT films show stable performance under >100% strain. This material synergistically combines high electrical conductivity, exceptional mechanical ductility, and patternability by printing, thus opening many new avenues toward next-generation wearable and epidermal electronics and bioelectronics."
} | 1,645 |
33500958 | PMC7805616 | pmc | 6,033 | {
"abstract": "The relationship between a reinforcement learning (RL) agent and an asynchronous environment is often ignored. Frequently used models of the interaction between an agent and its environment, such as Markov Decision Processes (MDP) or Semi-Markov Decision Processes (SMDP), do not capture the fact that, in an asynchronous environment, the state of the environment may change during computation performed by the agent. In an asynchronous environment, minimizing reaction time—the time it takes for an agent to react to an observation—also minimizes the time in which the state of the environment may change following observation. In many environments, the reaction time of an agent directly impacts task performance by permitting the environment to transition into either an undesirable terminal state or a state where performing the chosen action is inappropriate. We propose a class of reactive reinforcement learning algorithms that address this problem of asynchronous environments by immediately acting after observing new state information. We compare a reactive SARSA learning algorithm with the conventional SARSA learning algorithm on two asynchronous robotic tasks (emergency stopping and impact prevention), and show that the reactive RL algorithm reduces the reaction time of the agent by approximately the duration of the algorithm's learning update. This new class of reactive algorithms may facilitate safer control and faster decision making without any change to standard learning guarantees.",
"conclusion": "6. Conclusions RL algorithms are built on four main components: acting, observing, choosing an action, and learning. The execution of any of these components takes time, and while this may not affect synchronous discrete-time environments, it is a critical consideration for asynchronous environments, especially when task performance is proportional to the reaction time of the agent. An agent should never have to wait to take an action after receiving up-to-date observations . In this paper we present a novel reordering of the conventional RL algorithm which allows for faster reaction times. We present a simple sketch for algorithmic equivalence in synchronous discrete-time settings and show improved performance in an asynchronous continuous-time stopping task which is directly linked to agent reaction time. These results indicate that (1) reaction time is an important consideration in asynchronous environments, (2) the choice of when in a loop the RL agent should act affects an agent's reaction time, (3) reordering of the components of the algorithm as suggested here will not affect an agent's performance in synchronous discrete-time environments, (4) reactive algorithms reduce the reaction time, and thus improve performance, potentially also decreasing the time it takes for an agent to learn an optimal policy. This work, therefore, has wide potential application in real-world settings where decision making systems must swiftly respond to new stimuli.",
"introduction": "1. Introduction Reinforcement learning (RL) algorithms for solving optimal control problems are comprised of four distinct components: acting, observing, choosing an action, and learning. This ordering of components forms a protocol which is used in a variety of applications. Many of these applications can be described as synchronous environments where the state of the environment remains in the same state until the agent acts at which point the environment immediately returns its new state. In these synchronous environments, such as Backgammon (Tesauro, 1994 ) or classic control problems, it is not necessary to know the computation time to perform any of the protocol's components. For this reason, most reinforcement learning software libraries, such as RL-Glue (Tanner and White, 2009 ), BURLAP 1 or OpenAI gym 2 , have functions which accept the agent's action, and return the new state and reward immediately. These functions remain convenient for simulated environments where the dynamics of the environment can be computed easily (Sutton and Barto, 1998 ). However, unlike synchronous environments, asynchronous environments, also referred to as dynamic environments, do not wait for an agent to select an action before they change state (Kober et al., 2013 ; Pilarski et al., 2015 ; Russell and Norvig, 2016 ). The computation of RL protocol components (acting, observing, choosing an action, learning) takes time and an asynchronous environment will continually change state during this time (Degris and Modayil, 2012 ; Hester et al., 2012 ; Caarls and Schuitema, 2016 ). This can negatively affect the performance of the agent. If the agent's reaction time is too long, its chosen action may become inappropriate in the now changed environment. Alternatively, the environment may have moved into an undesirable terminal state. In this paper, we explore a very simple alternative arrangement of the reinforcement learning protocol components. We first investigate a way to reorder SARSA control algorithms so that they are able to react to the most recent observation before learning about the previous time step; we then discuss convergence guarantees of these reordered approaches when viewed in discrete time (following Singh et al., 2000 ). Then, we examine a asynchronous continuous-time robot task where the reaction times of agents affect the overall task performance—in this case, breaking or not breaking an egg with a fast-moving robotic arm. Finally, we present a discussion on the implementation of reactive algorithms and their application in related settings. 1.1. Related background The focus of most contemporary RL research is on action selection, representation of state, and the learning update itself; the performance impact of reaction time is considered less frequently, but is no less important of a concern (Barto et al., 1995 ). Several groups have discussed the importance of minimizing reaction time (Degris and Modayil, 2012 ; Hester et al., 2012 ; Caarls and Schuitema, 2016 ). Hester et al. noted that existing model-based reinforcement learning methods may take too much time between successive actions and presented a parallel architecture that outperformed traditional methods. Caarls and Schuitema extended this parallel architecture to the online learning of a system's dynamics (Caarls and Schuitema, 2016 ). Their learned model allowed for the generation of simulated experience which could be combined with real experience in batch updates. While parallelization methods may improve performance, they are computationally demanding. We propose an alternative approach when system resources are constrained.",
"discussion": "5. Discussion Our results indicate that rearranging the fundamental components of existing TD-control algorithms (act, observe, choose action, learn) has a beneficial effect on performance in asynchronous environments where task performance is reaction-time dependent. A reactive agent can perform better in these environments as it can act immediately following observations. As would be expected, this effect becomes especially prominent as the duration of learning operations increases. Although the current experimental design added a simulated delay to the learning update step, our results indicate that as the time between observing and acting grows, performance in these environments deteriorates, regardless of the source of these delays. As standard RL algorithms perform learning and state representation construction [e.g., tile-coding, (Sutton and Barto, 1998 ), deep neural networks, (Silver et al., 2016 ), etc.] between observing and acting, additional computation time is necessary. In asynchronous environments, as these steps become longer, the order of algorithmic components [acting, observing, choosing an action, and learning] becomes more critical. As we have shown, performance in asynchronous environments is inversely proportional to the total length of time between observations and acting. One alternate means of addressing delay-induced performance concerns may be to create a dedicated thread for each of the RL algorithm components (c.f., Caarls and Schuitema, 2016 ). We believe this is a promising area for continued research. As suggested, reactive algorithms in this work may have great utility when applied to single-thread computers as they do not require multiple threads so while the order of algorithmic components might seem at first like a minor implementation detail, it may prove critical when applied to these systems. Put more strongly, we believe that allowing an RL agent to learn an optimal ordering of its learning protocol or to interrupt learning components for more pressing computations are interesting subjects of future work. As a thought experiment, imagine an oracle-agent that has perfectly modeled its environment, knowing the outcome of every possible action. If this environment is asynchronous and provides more positive rewards for completing a task as quickly as possible, then, in order for this oracle-agent to maximize its reward, it should eliminate all computations which are not necessary as they delay the agent. Since it has perfectly modeled its environment, learning does not and will not improve its model. Moreover, if by predicting the state using its perfect model, the agent can achieve a perfect state prediction without observing, observation is also an unnecessary computation. Thus the oracle-agent can eliminate learning and observing and can simply act. Similar to the oracle-agent, some human experts, such as video-game speed runners and musicians, are sometimes able to perform their talents without actually observing the consequences of their actions because they have memorized a long sequence of optimal actions and can act out this sequence without needing to observe its results (Mallow et al., 2015 ; Talamini et al., 2017 ). By viewing the order of algorithmic components of learning algorithms as modifiable, an agent is freed to be able to find an optimal ordering of its learning protocol which may allow it to interrupt long lasting computations (e.g., analyzing an image) for more pressing computations (e.g., avoiding a pedestrian). The ability for an artificial agent to re-order its computational components could prove to be better utilized in the case of multi-threaded systems. Computationally limited systems that have the capacity for operating systems that support multithreading, such as TinyOS, have the ability to be more expressive in the way they structure their computation (Levis et al., 2005 ). Artificial agents taking advantage of the techniques found in multithreaded systems could schedule their own learning update via tasks, invoke hardware interrupts, and delay expensive learning computations dynamically."
} | 2,699 |
33478513 | PMC7819241 | pmc | 6,034 | {
"abstract": "Background Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. Main body of the abstract In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes. Short conclusion Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.",
"conclusion": "Conclusion Ligninolytic enzymes produced by fungi have been extensively exploited in a number of industries due to their versatile application, which derives from their lignin-based compounds catalytic oxidation properties. The large variety of lignin modifying fungal species and related ligninolytic enzyme consortium described in this review have been used in the production of biofuels, antibiotics, and fermented products, as well as in bioremediation and biomedical application as biosensors. The essential prerequisite for a successful use of fungal ligninolytic enzymes is their production in large quantities. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. Application of CRISPR-Cas system has shown great efficiency in discovery, activation, and editing of fungal genes/BGCs, and it holds a potential headway for production or enhancement of ligninolytic enzymes in large-scale. In addition, many newly available algorithms have been used to optimize CRISPR-Cas systems for editing specific loci or estimating potential off-target effects if used in fungal genomes. Further research could be directed towards improving the production of ligninolytic secretomes (or new ligninolytic genes) with desired attributes such as thermal, catalytic, or substrate adaptability. Various bacteria such as E. coli and Bacillus subtilis and filamentous fungi such as Aspergillus and Trichoderma species have been used as synthetic hosts for the expression of some LEs from white rot fungi. Yeasts such as S. cerevisiae, P. pastoris, K. lactis , and Y. lipolytica have been identified with good minicellulosomes, thus being good candidates for synthetic production of ligninolytic secretomes. Several studies have demonstrated heterologous expression in yeasts of ligninolytic enzymes from higher fungi. These re-engineered yeasts show adequate enzyme secretion with high tither values that do not affect negatively their cellular physiology. Yeast transformants in most cases showed better tolerance to oxidative stress, after the experiment, and ability to produce recombinant LEs in multiple folds, having better specification, quality, and reactivity as compared to the wild type. In addition, recombinant LEs can be easily characterized with molecular structure and studied for iron trafficking/hierarchy distribution in the cell."
} | 1,227 |
34195563 | PMC8233197 | pmc | 6,036 | {
"abstract": "Summary Electrotrophic microorganisms have not been well studied in extreme environments. Here, we report on the nitrate-reducing cathodic microbial biofilm from a haloalkaline environment. The biofilm enriched via electrochemical approach under 9.5 pH and 20 g NaCl/L salinity conditions achieved − 43.5 ± 7.2 μA / cm 2 current density and 49.5 ± 13.2 % nitrate reduction efficiency via partial and complete denitrification. Voltammetric characterization of the biocathodes revealed a redox center with − 0.294 ± 0.003 V ( vs. Ag/AgCl) formal potential putatively involved in the electron uptake process. The lack of soluble redox mediators and hydrogen-driven nitrate reduction suggests direct-contact cathodic electron uptake by the nitrate-reducing microorganisms in the enriched biofilm. 16S-rRNA amplicon sequencing of the cathodic biofilm revealed the presence of unreported Pseudomonas, Natronococcus, and Pseudoalteromonas spp. at 31.45 % , 11.82 % , and 9.69 % relative sequence abundances, respectively. The enriched nitrate-reducing microorganisms also reduced nitrate efficiently using soluble electron donors found in the lake sediments, thereby suggesting their role in N-cycling in such environments.",
"conclusion": "Conclusions The electrochemical cultivation resulted in the enrichment of a haloalkaliphilic nitrate-reducing microbial biofilm composed mainly of Pseudomonas, Natronococcus, and Pseudoalteromonas spp. at the cathode surface. The nitrate-reducing microorganisms in the enriched biofilm most likely followed the direct-contact electron uptake mechanism rather than mediated by redox shuttles and hydrogen to reduce nitrate under autotrophic conditions. An unknown redox-active moiety putatively involved in the electron uptake process was revealed by electrochemical analysis of the biocathodes. The enriched nitrate-reducing culture also grew faster by using soluble electron donor sources under heterotrophic conditions. By reporting on the haloalkaliphilic nitrate-reducing biofilm composed of several microbial groups under electroautotrophic conditions, this study expands the known habitats for both electrotrophs and nitrate reducers. Further work on the isolation and detailed characterization of dominant microorganisms for electroactivity and nitrate reduction is expected to broaden the diversity of both extreme electroactive and nitrate-reducing microorganisms.",
"introduction": "Introduction Electromicrobiology deals with the study of electrochemical interactions or extracellular electron transfer (EET) processes between microorganisms and solid-state electron donors or acceptors required to maintain respiratory activities and their implications in different environments ( Lovley, 2012 ; Nealson and Rowe, 2016 ). EET by a microbial cell or biofilm can be either inward or outward transfer of electrons from or to a solid-state electron donor or acceptor. Microorganisms possessing EET capabilities are termed electroactive microorganisms ( Nealson, 2017 ; Logan et al., 2019 ; Kiran and Patil, 2019 ). Bioelectrochemical systems (BESs) are used to explore different applications of EAMs and study their EET mechanisms. EAMs are categorized into two main types, namely exoelectrogens and electrotrophs. Exoelectrogens are capable of outward EET, that is, from cells to the solid-state terminal electron acceptors such as mineral oxides and electrodes to achieve respiration, whereas electrotrophs are capable of inward EET, that is, from the reduced minerals or electrodes to the cells to acquire energy ( Lovley, 2012 ; Chiranjeevi and Patil, 2020 ). Electrotrophs use the acquired electrons to grow and reduce substrates such as carbon dioxide, nitrate, sulfate, and heavy metals. The components involved in both direct and indirect EET processes have been studied and reported mainly for exoelectrogens but rarely for electrotrophs ( Rosenbaum et al., 2011 ; Shi et al., 2016 ; Liu and Li, 2020 ). For instance, Shewanella oneidensis MR-1 has been proposed to follow the reverse Mtr pathway of electron transfer for reductive metabolism ( Ross et al., 2011 ) and electron uptake from the cathode electrode ( Rowe et al., 2018 ). Several studies have reported on the electrotrophic mixed-culture microbial biofilms, grown with electron acceptors such as oxygen ( Rabaey et al., 2008 ), H + ions ( Aulenta et al., 2008 ), CO 2 ( Aryal et al., 2017 ), heavy metals, polyaromatic hydrocarbons, dyes, and so on ( Gregory and Lovley, 2005 ; Tandukar et al., 2009 ; Sharma et al., 2020 ; Fang et al., 2016 ). Notably, EAMs have been studied mostly from nonextreme environments, and only a few studies have reported the diversity of these microbes inhabiting extreme habitats ( Koch and Harnisch, 2016 ; Rowe et al., 2017 ; Logan et al., 2019 ; Yadav and Patil, 2020 ). In particular, electrotrophs from extreme habitats have been barely studied, limiting our understanding of EET-capable microbes and their role in element cycling under specific environmental conditions and their use for various BES applications. Moreover, electrotrophy is a unique energy conservation mode followed by some microbes in the soluble-electron-donor-depleted environments ( Logan et al., 2019 ). It calls for extensive research on microbial resource mining to understand the diversity and EET mechanisms of electrotrophic microorganisms from extreme habitats and explore new strategies to use the EET-capable extremophiles for niche-specific biotechnological applications ( Koch and Harnisch, 2016 ; Schröder and Harnisch, 2017 ). Nitrate-reducing microorganisms play a vital role in nitrogen cycling in different environments and can be used to bioremediate nitrate contaminated waters or environments. They are crucial for maintaining and removing the available fixed “N” in nitrate into N 2 via intermediates such as nitrite in natural environments ( Jenkins and Kemp, 1984 ; Codispoti and Christensen, 1985 ). Both mixed and pure microbial cultures capable of drawing electrons from the cathode have been reported at pH 7 condition ( Table 1 ). Exploring extreme haloalkaline habitats for nitrate-reducing microorganisms capable of electrotrophy could lead to identifying novel microorganisms and electron transfer mechanisms besides understating their role in N-cycling in the soluble-electron-donor-depleted environments. Thus, the present study aimed to enrich and investigate nitrate-reducing microorganisms at the cathode of BES from a haloalkaline environment of the Lonar Lake (located in Buldhana District, Maharashtra, India). The Lonar crater lake is a hypersaline soda lake rich in various nutrients and supports a wide diversity of haloalkaliphilic microorganisms ( Paul et al., 2016 ; Antony et al., 2013 ; Chakraborty et al., 2020 ). We first enriched the haloalkaliphilic nitrate-reducing microbial biofilm at the cathode of BES by using an electrochemical cultivation approach. We then tested the enriched biofilm for its nitrate-reducing capability with soluble electron donor sources, selected to match those found in the lake sediments. It was followed by a detailed characterization of the enriched microbial biofilm via electrochemical, microscopic, and 16S-rRNA amplicon sequencing-based analyses. Based on the obtained results, we discuss the electrotrophic nature of the cathodic biofilm and the environmental importance of the haloalkaliphilic microbial groups present in the enriched biofilm. Table 1 A comparative overview of the nitrate-reducing biofilms or microorganisms reported in bioelectrochemical systems S. No. Source of microorganisms Major experimental conditions Applied potential (V vs. Ag/AgCl) OR mode of operation Maximum current density/Voltage Carbon source Reduced product Nitrate removal efficiency (%) or rate Formal potential (V vs. Ag/AgCl) of the redox peaks during substrate turnover conditions Reference 1 Geobacter metallireducens pH 6.8, 30°C −0.5 V NR NaHCO 3 NO 2 − 90% NR Gregory et al., 2004 2 Thiobacillus denitrificans pH 7, 30°C −0.606 V −4 to −5 μA/cm 2 CH 3 COOH (Possible electron donor) NR NR NR Kato et al., 2012 3 Pseudomonas alcaliphila pH 7, 30°C −0.5 V −48.75 ± 1.25 μA/cm 2 Na 3 C 6 H 5 O 7 NR 72.40 ± 2.09% NR Su et al., 2012 4 Thiobacillus denitrificans pH 7, 30°C −0.705 V −3.28 μA/cm 2 NO 3 NR 75.62 ± 5.97% −0.515 V Yu et al., 2015 5 Thioclava electrotropha ElOx9 pH 6.5, 30°C 342 mM NaCl −0.483 V −2.45 ± 1.3 μA/cm 2 NaHCO 3 NO 2 − NR −0.277 ± 0.005 V Karbelkar et al., 2019 6 Mixed culture enriched from anaerobic sludge dominated by α-proteobacteria, β-proteobacteria, γ-proteobacteria and flavobacteria. pH 7, 30°C Fixed current (200 mA) NR NaHCO 3 N 2 via NO 2 − NR NR Park et al., 2006 7 Mixed community enriched from wastewater sludge dominated by Proteobacteria, Bacteroidetes, Actinobacteria, Planctomycetes, Firmicutes, and uncultured bacteria. pH 7, 22°C 0.05% NaCl OCV −18.80 ± 1.6 A/m 3 0.101 ± 0.009 V NaHCO 3 NR 35.21 ± 7.41% NR Chen et al., 2010 8 Mixed community enriched from sludge pH 7, 30°C 0.7 V by the DC power supply 138.39 μA/cm 2 NaHCO 3 NR 91% −0.2 V Kondaveeti and Min, 2013 9 Mixed community enriched from anaerobic sludge pH 7.2, 30°C Fixed current of 200 mA NR NaHCO 3 NR 99% NR Tong et al., 2013 10 Mixed community enriched from return sludge dominated by Proteobacteria pH 7.4, 30°C 0.7 V applied by the DC power supply NR NaHCO 3 NR 88% −0.13 V Kondaveeti et al., 2014 11 Mixed culture enriched from fresh water sediments and denitrifying biomass from sewage treatment plant, dominated by Betaproteobacteria , including Rhodocyclales and Burkholderiales. pH 7, 30°C −0.25 V and −0.35 V −210 μA/cm 2 and −320 μA/cm 2 NaHCO 3 NR 14-40% −0.18 V/−0.24 V and −0.45 V Gregoire et al., 2014 12 Mixed culture enriched from cathodic biofilm, dominated by Thiobacillus sp. pH 8, 22°C 0.05% NaCl −0.32 V −3.60 to −3.78 μA/cm 2 NaHCO 3 NR 34.1–54.1% −0.30 V and −0.70 V Pous et al., 2014 13 Mixed culture enriched from activated sludge dominated by Geobacter sp. pH 7, 22°C −0.508 V NR NaHCO 3 NR 532 mg N/m 2 /day −0.38 ± 0.034 V/−0.363 ± 0.033 V Pous et al., 2016 14 Mixed Community enriched from anaerobic sludge dominated by Shinella sp. and Alicycliphilus sp. pH 7, 25°C −0.905 V NR NaHCO 3 NO 2 − , N 2 and NH 3 3.5 mg/L/day NR Nguyen et al., 2016a 15 Mixed community enriched from anaerobic sludge dominated by Thiobacillus sp. and Paracoccus sp. pH 7, 25°C −0.905 V NR NaHCO 3 NR 322.6 mg/m 2 /day NR Nguyen et al., 2016b 16 Mixed community enriched from pharmaceutical wastewater pH 6.5–6.6 OCV 0.253 V (OCV) CH 3 COOH (electron donor) N 2 83% NR Nikhil et al., 2017 17 Mixed community enriched from previous enrichment reactor dominated by Acholeplasma and Azoarcus genera pH 7.5, 25°C Fixed current of 5–100 μA 0.73–0.224 V (OCV) NaHCO 3 NR 17% NR Ding et al., 2017 18 Mixed community enriched from Lonar Lake sediments dominated by Pseudomonas, Natronococcus , and Pseudoalteromonas spp. pH 9.5, 25°C 2% NaCl −0.3 V −43.5 ± 7.2 μA/cm 2 NaHCO 3 NO 2 − and N 2 49 . 5 ± 13 . 2 % − 0.724 ± 0.003 V and − 0.29 ± 0.003 V This study OCV: open-circuit voltage; NR: not reported.",
"discussion": "Results and discussion Chemical analysis of the sediment samples The pH and salinity of the sediment samples collected from three different sampling locations were 9.5 ± 0.2 and 14.3 ± 1.0 g/L, respectively ( Table S1 ). The salinity of the lake has been reported to vary in a range from 5 to 24 g/L owing to sampling and seasonal variations ( Sengupta and Bhandari, 1997 ; Borul , 2012 ; Jadhav and Mali , 2018 ). Based on these observations, the growth medium with a pH of 9.5 and salinity of 20 g/L was used to enrich nitrate-reducing microorganisms. Notably, the sediment samples were found to be rich in various soluble ions such as S O 4 2 − and N O 3 − ( Table S1 ), which can act as soluble electron acceptors in microbial respiratory processes. In particular, N O 3 − at about 222.4 ± 7.0 mg/L suggests the likely presence of nitrate-reducing microorganisms in this extreme habitat. Enrichment of the haloalkaliphilic nitrate-reducing microbial biofilm at the cathode The bioelectrocatalytic reduction current and nitrate concentration in the bulk phase were monitored to check the growth of nitrate-reducing microorganisms at the polarized cathode surface ( Figure 1 ). In the case of enrichment reactor R1, the reduction current started to increase after approximately 40 days ( Figure 1 A), suggesting a long start-up period, most likely owing to the time required by microorganisms to acclimatize to the challenging solid-state electron donor conditions. In subsequent batch cycles, the increase in the reduction current response was observed, which was accompanied by the decrease in the nitrate concentration. The abiotic-connected control showed no considerable reduction current response and no change in the nitrate concentration. Neither microbial growth nor change in nitrate concentration was observed in the biotic-unconnected control reactor ( Figure S1 ). These observations suggested the occurrence of microbial nitrate reduction process by utilizing cathodic electrons, i.e., the enrichment of nitrate-reducing microorganisms at the cathode surface. On completing the first batch cycle, the enriched biofilm in R1 was used as the microbial inoculum for inoculating reactors R2 ( Figure 1 B) and R3 ( Figure 1 C). The scrapping of some biomass from the cathode of R1 decreased the current density considerably in the second cycle, which, however, regained in the third cycle ( Figure 1 A). The enriched nitrate-reducing biofilm in R1 achieved a maximum current density of − 48.86 μ A / c m 2 with a corresponding nitrate removal efficiency of 57.8%. Figure 1 Chronoamperometry and nitrate/nitrite concentration profiles Bioelectrocatalytic current generation and nitrate reduction by the enriched electrotrophic microbial biofilms in reactors R1 (A), R2 (B), R3 (C), and R4 (D). Similar bioelectrocatalytic performance in the first batch cycle of R2 was observed but within 15 days ( Figure 1 B). The use of already enriched biofilm as an inoculum source resulted in a decrease in the start-up time in the current response. A clear relation between the decrease in nitrate concentration and an increase in the reduction current response was observed. The maximum current density and nitrate removal efficiency achieved in this case were − 42.74 μ A / c m 2 and 65.14 % in the second batch cycle, respectively. These data further confirmed the enrichment of microorganisms capable of reducing nitrate by using electrons from the cathode of BESs. In studies conducted at nonextreme pH and salinity conditions, improved nitrate removal efficiencies have been reported ( Table 1 ). For instance, Tong et al. reported 99% efficiency with a mixed microbial culture ( Tong et al., 2013 ). Similarly, Kondaveeti and Min also achieved a maximum nitrate reduction efficiency of 91% with the mixed culture ( Kondaveeti and Min, 2013 ). With a pure culture of Geobacter metallireducens, a maximum nitrate removal of 90% has been reported ( Gregory et al., 2004 ). The third reactor, R3, was run for five cycles; in which, the first three were with N 2 headspace and the rest two were conducted with helium (He) gas in the headspace. In this case, the maximum current density achieved was − 45.05 μ A / c m 2 with a corresponding nitrate removal efficiency of 39.24% along with a production of 42.56 mg/L (2.13 × 10 −4 number of moles of N O 2 − − N ) nitrite and 3.17 × 10 −4 number of moles of N 2 –N. The corresponding electron recovery for N O 2 − − N and N 2 –N was 16.49% and 61.45%, respectively. The nitrate removal efficiencies for the second cycle of R4 and the first cycle of R5 (operated under He gas headspace) were 39.38% and 52.44%, respectively. These values corresponded to 34.36 mg/L (2.18 × 10 −4 number of moles of N O 2 − − N ) and 30.05 mg/L (1.52 × 10 −4 number of moles of N O 2 − − N ) of nitrite formation in R4 and R5, respectively ( Figures 1 D and S2 ). In the same cycles, a maximum electron recovery of 74.61% in N 2 –N (3.02 × 10 −4 number of moles of N 2 –N) and 55.83% (6.10 × 10 −4 number of moles of N 2 –N) was achieved in R4 and R5, respectively. All the reactors were operated for at least two batch cycles by replenishing the spent medium with a fresh medium. Hence, the instant reduction current response in the subsequent cycles suggested that the bioelectrocatalytic activity (i.e., electron uptake from the cathode) was primarily because of the cathodic biofilm rather than microorganisms present in the bulk phase. The protein estimation via Bradford assay (∼0.007 mg/L protein concentration) and OD 600 revealed very little cell growth in the suspension. The protein content of the cathodic biofilm was estimated to be ∼0.8 mg/L. These data suggest that microorganisms in the biofilm contributed predominantly to the nitrate reduction process. No significant fluctuation in the pH (9.5 ± 0.29) of the medium was observed throughout the experiments. Moreover, ammonium ion concentration remained almost constant in each batch cycle in the medium, which confirms that no dissimilatory nitrate reduction to ammonium process occurred in the system. These data support that both partial and complete denitrification of nitrate to nitrite and nitrogen occurred in BESs. The trace amount of H 2 detected in the abiotic setup accounted for about 0.2–0.8% of the total current production of the biotic reactors. Two additional experiments were conducted to check and confirm the role of H 2 in the nitrate reduction process. In the first experiment conducted in the electrically unconnected bioelectrochemical reactor with the enriched cathodic biofilm and fed with H 2 as the only electron source, no decrease in nitrate concentration was observed ( Figure S3 A). However, about 26% consumption of H 2 was observed after six days of the batch experiment. In the second experiment, serum flasks with H 2 as the only electron source and nitrate as electron acceptor were set and inoculated with the enriched cathodic biofilm. Although OD 600 increased from the initial 0.035 to a maximum of 0.6, there was no decrease in the nitrate concentration in the serum flasks after four days of incubation ( Figures S3 B and S3C). The other key observation was about 23.6% consumption of H 2 by the microbes. The results from both experiments suggest that microorganisms that are not involved in the nitrate reduction reaction consumed H 2 . Electrochemical characterization of the haloalkaliphilic nitrate-reducing biofilm Cyclic voltammograms (CVs) were recorded to detect any redox-active moieties or components in the catholyte and the cathode surface at different conditions before and after the enrichment experiments. The representative CVs for R1, recorded before and after inoculation and during substrate turnover and nonturnover conditions, are shown in Figure 2 . Figure 2 Representative cyclic voltammograms recorded under different conditions for R1 (A) CVs at different conditions, (B) first derivative of the CV recorded under the substrate turnover condition, and (C) CV of the fresh electrode in a filtered spent medium of R1. The CV recorded during the substrate turnover conditions (i.e., in the presence of nitrate) showed a typical electrocatalytic curve ( Figure 2 A, red line trace ) and revealed the presence of two redox-active moieties with midpoint potentials of − 0.724 ± 0.003 V and − 0.294 ± 0.003 V \n vs . Ag/AgCl ( Figure 2 B). Redox-active moieties with similar formal potential were also observed in the CV recorded under the substrate nonturnover conditions (i.e., absence of nitrate) ( Figure 2 A, black dashed line trace). Based on the cathodic potential ( − 0.3 V ) that was applied for enriching the biofilm, it can be deduced that the redox-active moiety with a formal potential of − 0.294 ± 0.003 V might be involved in the microbial electron uptake process from the cathode for nitrate reduction. Attributing any role to the other moiety, with a low formal potential of − 0.724 ± 0.003 V , that is not involved in the actual microbial electron uptake process during CA experiments would be speculative. However, based on the literature on nitrate-reducing microorganisms in BESs, this particular redox component might be involved in the nitrite reduction process ( Pous et al., 2014 ). Similar CV behavior was also observed in the case of nitrate-reducing biocathodes of R2, R3, and R4 ( Figure S4 - I, II, and III). Such redox peaks have been reported previously for the nitrate-reducing biocathodes. For instance, Gregoire et al. and Kondaveeti and Min reported redox peaks with a formal potential of − 0.18 V and − 0.2 V at pH 7 (which are equal to − 0.328 V and − 0.348 V at pH 9.5), respectively ( Gregoire et al., 2014 ; Kondaveeti and Min, 2013 ). These are close to the redox peak with a formal potential of − 0.294 ± 0.003 V found in the case of the haloalkaliphilic biofilm in this study. Pous et al. reported a redox-active moiety at − 0.7 V at pH 8 (equals to − 0.789 V at pH 9.5) ( Pous et al., 2014 ). It is close to the second redox-active moiety observed at − 0.724 ± 0.003 V in our study. Some other redox peaks have also been reported for nitrate-reducing biofilms, as summarized in Table 1 . The CVs recorded with the fresh cathodes in the filtered spent medium of all reactors showed no redox peaks, thereby suggesting the absence of any soluble electron mediators or components in the medium ( Figures 2 C and S4 C- I, II and III). It also suggests that the nitrate-reducing biofilm enriched at the cathode surface most likely follows the direct mode for electron uptake and suggests its electrotrophic nature. A redox peak with a midpoint potential of approximately − 0.637 ± 0.004 V was observed in all the control CVs, that is, recorded before and after inoculation. It was most likely owing to the presence of a redox-active component in the medium or at the electrode surface. Hence, it is not considered as the redox-active component of the microbial biofilm grown or enriched at the cathode surface. Nitrate reduction by the enriched cathodic microbial biofilm with soluble electron donor sources The ability of the cathodic biofilm to grow by using the soluble electron sources was tested using citrate and acetate that are present in the lake sediments. Two sets of experiments were conducted with either He or N 2 in the serum flask headspace for each electron donor condition. An increase in the turbidity (i.e., OD 600 ) corresponding with nitrate reduction suggested the growth of haloalkaliphilic nitrate-reducing microorganisms with the soluble electron donor ( Figures 3 A, 3B, S5 A, and S5B). About 45.92 ± 9.73% of nitrate reduction and 85.93 ± 0.29% of citrate oxidation along with 6.35 × 10 −5 moles of N 2 production were observed at the end of the third batch cycle in He-sparged serum flasks. With acetate as the electron donor, the observed microbial growth in terms of OD 600 was lesser than citrate. About 75.59 ± 1.44% acetate oxidation and 93.12 ± 0.48% nitrate reduction along with 9.87 × 10 −5 moles of N 2 production were observed at the end of the third cycle in He-sparged serum flasks ( Figures 3 A and 3B). Nitrite concentrations increased for the first two days of each batch cycle and then subsequently decreased to zero at the end of the cycles. Similar growth and nitrate reduction trends were observed in the N 2 -sparged serum flasks ( Figures S5 A and S5B). Neither increase in OD 600 nor decrease in nitrate concentrations was observed in the biotic and abiotic controls ( Figures 3 C, 3D, S5 C, and S5D). These observations suggest that no nitrate reduction occurred under the abiotic condition and in the absence of an electron donor source for microbial cells in the biotic control. The OD data reveal a much shorter lag phase (about 24 h) than the growth under insoluble electron donor (i.e., cathode) and autotrophic conditions in electrochemical cultivation experiments. It suggests the slow growth of nitrate-reducing microorganisms under the electroautotrophic condition. The serum flask experiment observations suggest the role of enriched microorganisms in the cycling of both carbon and nitrogen elements under the haloalkaline growth conditions. Figure 3 Nitrate reduction profiles of the enriched culture with different electron donors Microbial growth (in terms of OD 600 ) and nitrate reduction profiles of the enriched nitrate-reducing culture over three batch cycles of the serum flask experiments (n = 3; with He gas in the headspace) with citrate (A) and acetate (B) as electron donors. The data of respective abiotic and biotic control experiments are presented in (C) and (D). Visualization of the microorganisms at the cathode surface and in the bulk phase of reactors The scanning electron microscopy (SEM) imaging revealed the presence of microbial cells at the cathode surface ( Figures 4 A and 4B) and in the bulk phase ( Figures 4 C and 4D) of bioelectrochemical reactors. In addition, in the suspension of the serum flasks, oval-shaped cells were observed ( Figures 4 E and 4F). These results confirm the growth of microorganisms in the experiments conducted under both insoluble and soluble electron donor conditions. The biofilm formation at the cathode surface was, however, not uniform and instead was in patches ( Figure 4 A). Nevertheless, the microbial growth at the cathode surfaces, along with the electrochemical data, confirms the enrichment of haloalkaliphilic nitrate-reducing biofilm. The weight percentage data of FEG-SEM-EDS revealed the absence of any large elements on the surface of biocathodes and abiotic cathode except for the presence of elements, mainly C and O (at 29.17 wt.% and 8.16 wt.%, respectively) and trace amounts of elements such as Al and Ir (at 0.63 wt.% and 0.35 wt.%, respectively) ( Figure S6 ). These might be involved in the catalysis of H 2 evolution at the cathodic surface. Figure 4 Visualisation of the microorganisms via scanning electron microscopy Representative SEM images for the enriched nitrate-reducing microorganisms at the cathode surface (A and B) and in the bulk phase (C and D) of BESs. The images in panels (E) and (F) are for the nitrate-reducing microbial cells grown with citrate as a soluble electron donor in serum flasks. Microbial community composition of the enriched nitrate-reducing electrotrophic biofilm The 16S-rRNA-amplicon-sequencing-based analysis of the enriched cathodic biofilm revealed the presence of species belonging to Pseudomonas, followed by Natronococcus, and Pseudoalteromonas at 31.45 % , 11.82 % , and 9.69 % relative sequence abundances, respectively ( Figure 5 ). In the Lonar lake sediments, that is, the inoculum source, Pseudomonas , Natronococcus, and Pseudoalteromonas genera were found to be at only 0.264%, 1.815%, and 0.762% relative sequence abundances, respectively ( Figure 5 ). These data suggest that the enriched microbial biofilm through the electrochemical cultivation approach led to selecting a few groups at the cathode capable of growing on the insoluble electron donor source. The most abundant genera present in the cathodic biofilm have been reported for their nitrate-reducing metabolic capabilities in different experimental conditions ( Tindall et al., 1984 ; Mulla et al., 2018 ; Su et al., 2012 ). In particular, microorganisms belonging to the Pseudomonas genus are known for their electroactivity ( Logan et al., 2019 ). For instance, the pure cultures of Pseudomonas, namely, Pseudomonas alcaliphila ( Su et al., 2012 ) and Pseudomonas aeruginosa ( Jia et al., 2017 ) have been reported to reduce nitrate at pH 7 ( Table 1 ). As far as the extreme habitats are concerned, this is the first study that reports on the haloalkaliphilic Pseudomonas spp. that most likely possesses the nitrate-reducing capability. Figure 5 16S rRNA-amplicon-sequencing-based microbial community composition in the Lonar lake sediments used as the inoculum source (sediment inoculum) and the enriched biofilm at the cathode surface (biocathode) Others represent the microbial communities present at < 1% relative sequence abundance. A few Natronococcus species have been reported for nitrate-reducing capability but not for electroactivity or electrotrophic properties. For instance, haloalkaliphilic Natronococcus occultus and Natronococcus amylolyticus have been reported to grow optimally at 9.5 pH and 20%–22% salinity by reducing nitrate to nitrite using glucose, ribose, or sucrose as the carbon and electron source ( Tindall et al., 1984 ; Kanai et al., 1995 ). Both species have been isolated from the East African soda lake, Lake Magadi, via conventional cultivation approaches. Another species named Natronococcus roseus, isolated from Chagannor Lake, China, has also been reported to reduce nitrate ( Corral et al., 2013 ). The presence of Natronococcus in the enriched cathodic biofilm in our study suggests that this genus might possess the traits that facilitate their growth under electrotrophic conditions. Similarly, several Pseudoalteromonas spp. , such as Pseudoalteromonas arabiensis, Pseudoalteromonas lipolytica, and Pseudoalteromonas prydzensis, isolated from the oxygen-depleted regions of Arabian Sea, have been reported to be involved in nitrate and nitrite reduction processes ( Mulla et al., 2018 ). They also possess various genes involved in the nitrogen cycle ( Cai and Jiao, 2008 ). However, they have not been reported for nitrate reduction process using the insoluble electron donors such as polarized cathodes. Hence, the enrichment of Pseudoalteromonas in the cathodic biofilm in this study suggests its probable electrotrophic nature. Actinobacteria and Vibrio, with a relative sequence abundance of 5.4% and 4.4%, respectively, have been reported to act as nitrate reducers in acidic or neutral and estuarine environments ( Palmer and Horn, 2015 ; Zenova et al., 2011 ; Macfarlane and Herbert, 1982 ). Thus, these can also be implicated in nitrate reduction in BESs. Some other microbial communities, namely, Geoalkalibacter, Halalkalicoccus, and Azoarcus that are known to reduce nitrate, were also present in the enriched electrotrophic EABs, but at very low relative sequence abundances of <0.00004%. Among Halalkalicoccus genus, Halalkalicoccus tibetensis (haloalkaliphile) and Halalkalicoccus paucihalophilus (halophile) isolated from the Lake Zabuye and Lop Nur region, China, respectively, are capable of reducing nitrate to nitrite. Finally, Azoarcus species strain PA01 T has also been reported to use nitrate as a terminal electron acceptor ( Junghare et al., 2015 ). Methanobacterium, Anaerovibrio, and Lachnospiraceae UGC-008 with relative sequence abundances of 2.2%, 1.4%, and 1.2%, respectively, were found in the enriched biofilm. However, their role has not been reported for the nitrate reduction process, so they might be involved in syntrophic relationships in the mixed microbial biofilm for sharing electron donors or acceptors. For instance, the traces of electrochemically produced H 2 in BESs were not used by the nitrate-reducing microorganisms but most likely supported the growth of methanogens in the enriched biofilm. In addition to these genera, an uncultured bacterium was found in the cathodic biofilm at a relative sequence abundance of 8.8%. Because its identity is not clear, its role cannot be discussed. Role of enriched microorganisms in the cathodic electron transfer and nitrate reduction process Many Pseudomonas spp. can produce mediators such as pyocyanin and phenazine-1-carboxamide, which are known to enhance their electron transfer process and thereby current production efficiency of bioelectrochemical systems ( Rabaey et al., 2005 ; Venkataraman et al., 2010 ). A few studies have reported certain Pseudomonas spp. to produce phenazine compounds in either alkaline or saline conditions ( Zhang et al., 2011 ; Patil et al., 2016 ; Yuan et al., 2020 ). For instance, Sahoo et al. reported P. aeruginosa to produce phenazine compounds at saline and moderately alkaline (pH 8) conditions ( Sahoo et al., 2019 ). The high-resolution mass spectroscopy analysis of the cathodic biofilm and the spent medium revealed the absence of both pyocyanin and phenazine-1-carboxamide in our study ( Figures S7 and S8 ). These results confirm the CV observations on the lack of any soluble mediators in the bulk phase. These data and the lack of hydrogen-driven nitrate reduction together suggest that the electron transfer was biofilm-associated and most likely via direct electron transfer in the case of nitrate-reducing microorganisms. One plausible way of electron transfer among microorganisms in a mixed community is direct interspecies electron transfer ( Semenec et al., 2018 ; Li et al., 2020 ; Mostafa et al., 2020 ). It can involve uptake of cathodic electrons by a non-nitrate-reducing microbial group and then their transfer to nitrate-reducing microbes, that is, syntrophic exchange of electrons among different microbial groups to fulfill their growth or metabolic requirements. However, this possibility needs to be confirmed through dedicated experiments with the pure cultures of microbes present in high relative sequence abundance in the nitrate-reducing cathodic biofilm. The Lonar lake sediments contain nitrate at a considerable concentration (222.4 mg/L). In addition, organic and inorganic electron donor sources such as acetate, citrate, Fe, and Mn are present in the lake sediments. Although present at the low relative sequence abundance in sediments, the capability of different microbial groups such as Pseudomonas, Natronococcus and, Pseudoalteromonas present in the enriched culture to reduce nitrate to nitrite and N 2 \n via partial or complete denitrification processes using soluble electron donor sources suggests their possible role in N cycling in the haloalkaline environment. The nitrate reduction by the enriched culture with cathode as the insoluble electron donor also infers its probable capability to use reduced Fe minerals as the source of electrons in the sediments ( Beller et al., 2013 ; Yu et al., 2015 ; Liu et al., 2019 ). However, the proposed role of enriched microbes in the cycling of N, C, and Fe elements needs to be investigated in dedicated experiments with the pure culture isolates. To summarize, none of the enriched microbial groups in the cathodic biofilm, with the highest relative sequence abundances observed in this study, were reported earlier for nitrate reduction under highly saline-alkaline conditions. The following observations suggest the electrotrophic nature of the enriched nitrate-reducing microorganisms in the cathodic biofilm. • Most electric current response was observed only in the test reactors with cathodic microbial biofilm and not in the control reactors. • Soluble redox mediators, in particular, the most commonly produced ones by Pseudomonas spp., namely pyocyanin and phenazine-1-carboxamide, were not detected in the reactors. • No artificial redox mediator was supplied in the medium. • The trace amount of electrochemically produced H 2 in the abiotic experimental setup contributed only to 0.2%–0.8% of the total current density produced in the biotic reactors. Moreover, it was not used as the source of electrons for nitrate reduction by the enriched microorganisms in the cathodic biofilm. Conclusions The electrochemical cultivation resulted in the enrichment of a haloalkaliphilic nitrate-reducing microbial biofilm composed mainly of Pseudomonas, Natronococcus, and Pseudoalteromonas spp. at the cathode surface. The nitrate-reducing microorganisms in the enriched biofilm most likely followed the direct-contact electron uptake mechanism rather than mediated by redox shuttles and hydrogen to reduce nitrate under autotrophic conditions. An unknown redox-active moiety putatively involved in the electron uptake process was revealed by electrochemical analysis of the biocathodes. The enriched nitrate-reducing culture also grew faster by using soluble electron donor sources under heterotrophic conditions. By reporting on the haloalkaliphilic nitrate-reducing biofilm composed of several microbial groups under electroautotrophic conditions, this study expands the known habitats for both electrotrophs and nitrate reducers. Further work on the isolation and detailed characterization of dominant microorganisms for electroactivity and nitrate reduction is expected to broaden the diversity of both extreme electroactive and nitrate-reducing microorganisms. Limitations of study This study reports on the haloalkaliphilic nitrate-reducing microorganisms, which are slow growing under electroautotrophic conditions. The redox center involved in the electron uptake process cannot be attributed to any specific microbial group or component because of the mixed culture biofilm. In addition, specific electrotrophic microorganisms cannot be pointed out conclusively in the enriched mixed biofilm."
} | 9,319 |
28261048 | PMC5309244 | pmc | 6,040 | {
"abstract": "Here we provide the state-of-the-art of bioelectronic interfacing between biological neuronal systems and artificial components, focusing the attention on the potentiality offered by intrinsically neuromorphic synthetic devices based on Resistive Switching (RS). Neuromorphic engineering is outside the scopes of this Perspective. Instead, our focus is on those materials and devices featuring genuine physical effects that could be sought as non-linearity, plasticity, excitation, and extinction which could be directly and more naturally coupled with living biological systems. In view of important applications, such as prosthetics and future life augmentation, a cybernetic parallelism is traced, between biological and artificial systems. We will discuss how such intrinsic features could reduce the complexity of conditioning networks for a more natural direct connection between biological and synthetic worlds. Putting together living systems with RS devices could represent a feasible though innovative perspective for the future of bionics.",
"conclusion": "Conclusions After being theorized 40 years ago, it is only in the last decade that the fabrication of intrinsically neuromorphic devices was demonstrated. Following this milestone, in the latest years relevant efforts in the scientific community was directed toward the development of new materials for RSDs as well as theoretical algorithms for their use. One of the most promising applications of RSDs is in developing neuronal networks. On the other hand in the recent years it was possible to grow neurons over artificial substrates and new methodologies for the activity recording allowed the study of signals in neuronal networks and direct interaction with bio-artificial circuits, with a specific care in the simultaneous recording of signals from a complex network of neurons, in place of a single and isolated cell. Coupling RSDs with neuronal networks is still a distant objective. Nowadays we are at the edge of a new era where it will be possible to conceive and develop systems with reliable electrical interfaces between the brain and RSD-based neuronal networks, with the possibility of integrating them in wearable and comfortable devices. Neural prosthetics will be interfaced directly without any computer translation and used to fight serious neurological conditions resulting from disease, aging, or injury. And what about enhancing cognitive capacities of healthy people? The logical next step.",
"introduction": "Introduction The brain is the most powerful and complex known computational system. A recent work evaluates the memory capacity of the human brain to be in the order of 10 15 Bytes (Bartol et al., 2016 ). Unlike the hardware and software of a machine, the mind and brain are not distinct entities, feature that resembles the so called firmware . How could we represent a neuronal synapse, a complex structure containing hundreds of different proteins with a single line of code? We still do not know the detailed circuitry of any region of the brain well enough to reproduce its structure and, as a consequence, its behavior (Brooks et al., 2012 ). The technological roadmap toward integration of synthetic and biological functions was described in the past as cybernetics, in a definition given by N. Wiener (Wiener, 1961 ) that recalls ancient Greek κ υ β ε ρ ν η τ ι κ ή τ έ χ ν η , the art of the pilot. This definition moves from the hypothesis that there is a substantial analogy between self-regulation mechanisms in living beings and machines, based on information flow and feedback/closed loop. Putting the focus on silicon microdevices, we should trace a boundary between (Breslin and O'Lenskie, 2001 ). extrinsic neuromorphic systems , based on CMOS circuits that enable processing of information as occurs naturally in biological brains, including silicon based artificial synapses and artificial neurons sorted in neural networks, that are outside the scopes of this review; intrinsic neuromorphic systems , artificial synapses or arrays of elements that inherently possess key figures such as plasticity, non-linearity, spiking processing capabilities. Giant projects / frameworks, such as the DARPA SyNAPSE program (Systems of Neuromorphic Adaptive Plastic Scalable Electronics) and the EU Human Brain Project deal with standard extrinsic systems and therefore are outside the scopes of this Perspective. Their paradigm is reproducing the configurational complexity by emulating a simplified physical model in an extremely high number of elements (at least 10 10 neurons and 10 14 synapses)."
} | 1,146 |
37367613 | PMC10301997 | pmc | 6,042 | {
"abstract": "Arbuscular mycorrhizal fungi (AMF) play key roles in enhancing plant tolerance to heavy metals, and iron (Fe) compounds can reduce the bioavailability of arsenic (As) in soil, thereby alleviating As toxicity. However, there have been limited studies of the synergistic antioxidant mechanisms of AMF ( Funneliformis mosseae ) and Fe compounds in the alleviation of As toxicity on leaves of maize ( Zea mays L.) with low and moderate As contamination. In this study, a pot experiment was conducted with different concentrations of As (0, 25, 50 mgꞏkg −1 ) and Fe (0, 50 mgꞏkg −1 ) and AMF treatments. Results showed that under low and moderate As concentrations (As25 and As50), the co-inoculation of AMF and Fe compound significantly increased the biomass of maize stems and roots, phosphorus (P) concentration, and P-to-As uptake ratio. Moreover, the co-inoculation of AMF and Fe compound addition significantly reduced the As concentration in stem and root, malondialdehyde (MDA) content in leaf, and soluble protein and non-protein thiol (NPT) contents in leaf of maize under As25 and As50 treatments. In addition, co-inoculation with AMF and Fe compound addition significantly increased the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the leaves of maize under As25 treatment. Correlation analysis showed that stem biomass and leaf MDA content were very significantly negatively correlated with stem As content, respectively. In conclusion, the results indicated that the co-inoculation of AMF and Fe compound addition can inhibit As uptake and promote P uptake by maize under low and moderate As contamination, thereby mitigating the lipid peroxidation on maize leaves and reducing As toxicity by enhancing the activities of antioxidant enzymes under low As contamination. These findings provide a theoretical basis for the application of AMF and Fe compounds in the restoration of cropland soil contaminated with low and moderate As.",
"conclusion": "5. Conclusions Under the stress of medium to low arsenic contamination, inoculation with arbuscular mycorrhizal fungi and the addition of iron compounds can synergistically promote phosphorus uptake while inhibiting arsenic uptake, leading to an increase in the phosphorus-arsenic uptake ratio and ultimately promoting maize growth. The synergistic effect of exogenous AMF and iron can alleviate lipid peroxidation in maize leaves under medium to low arsenic stress, reduce the soluble protein content and non-protein thiol content in low arsenic-polluted maize leaves, and enhance the activities of hydrogen peroxide, peroxidase, and superoxide dismutase in low arsenic-polluted maize leaves. Thus, exogenous AMF and iron compounds can synergistically promote the antioxidant defense system to counter arsenic toxicity in maize. In conclusion, AMF and iron compounds can be used to effectively improve plant tolerance to As by synergistically regulating plant physiological and biochemical mechanisms. The results of this work provide a theoretical basis for the remediation of medium to low arsenic-polluted agricultural soils.",
"introduction": "1. Introduction Arsenic (As) is commonly present in nature and is recognized as a carcinogen and environmental pollutant [ 1 , 2 , 3 ]. Arsenic in soil can accumulate in crops and migrate into lake water and groundwater, affecting crop yield and also causing serious harm to human health and local ecosystems [ 4 , 5 , 6 ]. When crops absorb excessive amounts of arsenic from the soil, they are subject to arsenic toxicity, which can manifest as inhibited root growth, stunted plant growth and development, and even death, resulting in decreased crop yield and affecting food security [ 7 , 8 , 9 ]. Physiologically, the toxicity of arsenic leads to the accumulation of reactive oxygen species in plant cells [ 10 ]. When the reactive oxygen species produced exceed the capacity of the reactive oxygen clearance system, plants experience inhibited chlorophyll synthesis, membrane lipid peroxidation, and damage to DNA, proteins, and some biomolecules [ 11 ]. The concentration of arsenic in agricultural soils in South and East Asia has been reported to range from 0 to 6402 mg·kg −1 , indicating severe arsenic pollution [ 12 , 13 ]. Maize ( Zea mays L.) is the most widely cultivated cereal worldwide, however, a study has shown that in countries with high maize yields, such as China, Argentina, India, and Mexico, the soil arsenic concentration greatly exceeds the global average soil background value (10.0 mg·kg −1 ) [ 14 ]. Therefore, the management of arsenic-polluted soil on farmland has become an important focus in environmental science [ 15 , 16 , 17 , 18 ]. Plants have evolved a series of defense mechanisms to resist external environmental stressors. When plants are subjected to arsenic stress, enzymes that synthesize plant chelators (PCs) can be activated. The thiol group on PCs can form a complex with reduced trivalent arsenic, which can be transported into the vacuole for storage to alleviate arsenic toxicity. However, the synthesis of PCs consumes the plant antioxidant glutathione, thus reducing the availability of glutathione to remove reactive oxygen species [ 19 ]. When plants are subjected to oxidative stress caused by excessive reactive oxygen species, they activate their non-enzymatic antioxidant system (including glutathione, ascorbic acid, carotenoids, and soluble proteins) and enzymatic antioxidant system (including SOD, POD, CAT, glutathione reductase, and glutathione-S-transferase) [ 20 , 21 ]. However, when the amount of arsenic entering the cell exceeds a certain limit, the activities of the components of these antioxidant enzyme and non-enzymatic systems will be inhibited, thereby causing harm to plant cells. Previous studies have shown that soil arsenic bioavailability and plant toxicity are related to the concentration and form of arsenic in the soil, plant species, and soil properties such as the content of iron oxides, redox potential, pH value, and phosphorus (P) content [ 22 , 23 ]. Numerous studies [ 24 , 25 , 26 , 27 ] have indicated that the inoculation of arbuscular mycorrhizal fungi (AMF) can have significant effects on plants, including increased absorption and utilization of mineral nutrients, altered uptake and transport of heavy metals, and alleviation of the adverse effects of heavy metal stress, thereby helping to improve the tolerance of host plants to heavy metals. Specifically, AMF colonization can enhance the arsenic resistance of either resistant or non-resistant plants grown in arsenic-contaminated soils by reducing arsenic biotoxicity [ 28 , 29 , 30 ]. AMF colonization can effectively enhance arsenic extraction for phytoremediation of heavily polluted soils, as demonstrated for the hyperaccumulator fern plant Pteris vittata [ 31 , 32 ]. Additionally, some studies have shown that there is a high affinity between iron and arsenic in soil [ 33 , 34 , 35 , 36 ]. Arsenic entering the soil can be adsorbed specifically or non-specifically onto the surface of iron oxides or hydroxides in the soil to form insoluble precipitates to alleviate the toxicity of arsenic to plants. In recent years, both domestic and international researchers have begun to investigate the synergistic effects of AMF and iron compounds and the effects on arsenic-contaminated plants [ 37 ]. Using a pot experiment, our research group [ 38 ] studied the combined effects of AMF and iron tailings to enhance plant resistance to arsenic by increasing the absorption of phosphorus and iron under moderate arsenic stress. However, there have been no reports on how the synergistic action of AMF and iron compounds alters the physiological and biochemical resistance mechanisms of leaf tissues. To address this, we employed a pot experiment to investigate the influences of different dosages of exogenous iron and AMF inoculation on physiological and biochemical indicators of maize leaves in soils contaminated with varying degrees of arsenic. The aim of this work was to elucidate the physiological and biochemical mechanisms by which AMF inoculation and exogenous iron compounds mitigate the phytotoxic effects of arsenic contamination. Specifically, we aimed to determine: (1) whether AMF and iron can synergistically regulate arsenic uptake in maize plants; (2) whether AMF and iron can synergistically alleviate the degree of membrane lipid peroxidation and reduce non-enzymatic antioxidant content of maize leaves and synergistically enhance antioxidant enzyme activities in maize leaves under arsenic stress; and (3) the plant response to different concentrations of AMF and iron under arsenic stress. We hypothesized that (1) either AMF or iron application alone would promote maize growth and increase phosphorus uptake compared to the control; and (2) AMF and iron would synergistically alleviate the degree of membrane lipid peroxidation, reduce the non-enzymatic antioxidant content of maize leaves, and synergistically enhance antioxidant enzyme activity in maize leaves under arsenic stress.",
"discussion": "4. Discussion 4.1. Effects of AMF Inoculation and Iron Supplementation on Membrane Lipid Peroxidation in Maize Leaves This study showed increased MDA content in maize leaves with higher levels of arsenic application ( p < 0.05). Hartley-Whitaker et al. [ 10 ], however, tested the arsenic-resistant genotype of velvet grass and found no significant changes in membrane lipid peroxidation under different arsenic concentrations. In contrast, in the non-resistant genotype, exposure to arsenic rapidly increased membrane lipid peroxidation in plant roots, illustrating that the addition of arsenic causes the accumulation of active oxygen (ROS) and exacerbates lipid peroxidation in non-resistant plant cells. During normal plant growth, ROS production and removal are in a dynamic balance, and the low free radical concentration will not cause plant damage. However, under arsenic contamination stress, the reduction of pentavalent arsenic to trivalent arsenic in plant cells disrupts the balance of ROS production and elimination, leading to ROS accumulation [ 47 , 48 ]. When the accumulation of ROS exceeds a certain concentration, it causes the oxidation of unsaturated fatty acids in cell membrane lipids in a process known as membrane lipid peroxidation. This process produces MDA, and the MDA content reflects the degree of lipid peroxidation. In this work, AMF inoculation and iron supplementation both reduced the MDA content in maize leaves for plants grown in arsenic-contaminated soil. This is related to the addition of iron and the inoculation of AMF to reduce the accumulation of arsenic in maize shoots. After AMF treatment, specific sites in the extra-root mycelium and spores of AMF can bind to heavy metal ions in the soil, thus immobilizing them in the mycelium and limiting their transfer to the host plant [ 49 ]. This suggests that AMF can store the heavy metal ions absorbed from the soil in the fungal structure and thus reduce the extent of plant damage. The membrane lipid structure of plant cells is protected. 4.2. Effects of AMF Inoculation and Iron Supplementation on the Contents of NPT and Soluble Proteins in Maize Leaves Phytochelatins (PCs) are a type of cysteine-rich peptide [ 50 ]. Heavy metal pollutants, such as copper, arsenic, cadmium, and zinc, can induce the synthesis of PCs [ 51 , 52 ] in plants that can act as detoxifying agents by chelating heavy metals with their thiol groups. Previous studies have shown that plant non-protein thiols (NPTs) are composed mainly of PCs, so they can be measured to indirectly reflect PC content [ 53 ]. In this study, the addition of arsenic increased the NPT content in maize leaves treated with NM, and AMF inoculation resulted in a trend of first decreasing and then increasing the NPT content in maize leaves with arsenic addition. Both AMF inoculation and iron supplementation reduced the NPT content in maize leaves treated with NM under low arsenic pollution. These results suggest that the addition of arsenic to soil may induce maize PC synthesis and thus increase the NPT content in leaves, with the medium arsenic treatment showing a relatively higher NPT content. However, the inoculation of AMF and iron supplementation significantly reduced the total arsenic content in maize plants under low arsenic pollution, alleviating arsenic toxicity and decreasing the NPT content in leaves. Soluble protein is one of the important components of the non-enzymatic antioxidant system [ 54 ], participating in cell osmotic regulation and also directly binding to heavy metal ions through its abundant hydroxyl, carboxyl, amino, aldehyde, and phosphate groups. This binding can reduce the interference of heavy metal ions with physiological metabolic processes in cells and decrease the damage to plant macromolecules such as DNA to improve plant growth [ 55 ]. In this study, with the increasing addition of arsenic, the content of soluble protein in maize treated with the NM-Fe0 combination also increased. This suggests that arsenic stress may induce protein production, and AMF inoculation may weaken the antioxidant function of soluble protein by enhancing plant arsenic resistance, resulting in a decrease in the soluble protein content. AMF inoculation can alter the biomass and root structure of plants under conditions of heavy metal contamination. This affects the uptake and translocation of heavy metals by the host, explaining one way that AMF treatment induces plant detoxification [ 56 ]. 4.3. Effects of Inoculating AMF and Iron Supplementation on Antioxidant Enzyme Activities in Maize Leaves When plants are subjected to abiotic stress, excessive ROS accumulation can occur, leading to oxidative damage. SOD, POD, and CAT are important antioxidant enzymes in plants. Despite having low activity under normal growth conditions, these enzymes can scavenge ROS, thereby preventing damage to the plant cell membrane structure. Arsenic-induced oxidative stress results in ROS accumulation within the plant, triggering activation of the endogenous antioxidant enzyme system (including SOD, POD, and CAT) and the increased antioxidant enzyme activities [ 57 ] scavenge excess ROS and protect against oxidative damage. SOD mainly functions to eliminate O 2 − , generating non-toxic O 2 and less toxic H 2 O 2 . CAT mainly scavenges H 2 O 2 generated by oxidative enzymes such as superoxide dismutase (SOD), glycolate oxidase, and urate oxidase, and POD helps CAT remove excess H 2 O 2 and other peroxides [ 58 ]. In this study, with the increase in arsenic level, the CAT activity in NM-Fe0-treated maize leaves increased, but at 50 mg·kg −1 of arsenic addition to the soil, both POD and SOD activities decreased significantly. The CAT activity in AMF-treated plants first increased and then decreased, indicating that the accumulation of arsenic may have caused oxidative stress and increased the activity of oxidases in the plants. However, the decrease in CAT activity in the As50+M treatment, and the decrease in POD activity and SOD specific activity in the As50-M treatment might reflect too much accumulated arsenic. This result is partly consistent with the conclusion of Mascher et al. [ 20 ] that SOD and POD activities in the aboveground part of Red Clover first increased and then decreased with increasing levels of arsenic. The results of this study show that under high arsenic stress, AMF inoculation can induce POD and SOD activities in maize leaves, which is consistent with the results reported by Zhan et al. [ 59 ]. In soil polluted with heavy metals, AMF can alleviate the toxicity of plants to heavy metals by enhancing the antioxidant defense capacity of leaves and the absorption of P by roots. Studies have shown that after AMF inoculation, plants reduce oxidative stress by enhancing the regulatory capacity of plant antioxidant systems and osmotic adjustment systems [ 60 ]. In addition, under arsenic addition, the combined treatment of iron supplementation and AMF inoculation can increase the CAT, POD, and SOD activities in maize leaves. This indicates that iron and AMF synergistically enhance the antioxidant defense system to improve the resistance of maize to arsenic pollution stress."
} | 4,074 |
31105660 | PMC6492693 | pmc | 6,043 | {
"abstract": "Microbial dissimilatory sulfate reduction to sulfide is a predominant terminal pathway of organic matter mineralization in the anoxic seabed. Chemical or microbial oxidation of the produced sulfide establishes a complex network of pathways in the sulfur cycle, leading to intermediate sulfur species and partly back to sulfate. The intermediates include elemental sulfur, polysulfides, thiosulfate, and sulfite, which are all substrates for further microbial oxidation, reduction or disproportionation. New microbiological discoveries, such as long-distance electron transfer through sulfide oxidizing cable bacteria, add to the complexity. Isotope exchange reactions play an important role for the stable isotope geochemistry and for the experimental study of sulfur transformations using radiotracers. Microbially catalyzed processes are partly reversible whereby the back-reaction affects our interpretation of radiotracer experiments and provides a mechanism for isotope fractionation. We here review the progress and current status in our understanding of the sulfur cycle in the seabed with respect to its microbial ecology, biogeochemistry, and isotope geochemistry.",
"introduction": "Introduction The sulfur cycle of marine sediments is primarily driven by the dissimilatory sulfate reduction (DSR) to sulfide by anaerobic microorganisms (e.g., Jørgensen and Kasten, 2006 ). This process links the complex food web of organic matter degradation to the terminal organic carbon oxidation to CO 2 . Most of the sulfide is ultimately reoxidized back to sulfate, via diverse sulfur intermediates, by geochemical or microbial reactions that involve oxygen, nitrate, manganese [Mn(IV)], iron [Fe(III)], and other potential oxidants (e.g., Rickard, 2012 ). A fraction of the sulfide precipitates with iron and other metals or reacts with organic matter and is buried deeply into the seabed. The microbial sulfur transformations affect the isotopic composition of sulfate and sulfides and the resulting isotope fractionation is thereby diagnostic for both process rates and pathways of the sulfur cycle (e.g., Canfield, 2001 ). We here review recent progress and selected aspects of these processes with emphasis on the interactions between microbial communities and the ambient sediment geochemistry. The processes are discussed with respect to their rates and pathways. We focus on fine-grained continental shelf sediments and do not discuss advective ecosystems such as cold seeps or hot springs or the low-energy ecosystems of the deep sea. Most examples are taken from coastal marine sediments of the Baltic Sea region. The cited data thereby provide a consistent picture of how the sulfur cycle may function in a specific seabed. With respect to the diversity and physiology of the respective microorganisms we refer to recent reviews (e.g., Finster, 2008 ; Muyzer and Stams, 2008 ; Knittel and Boetius, 2009 ; Rabus et al., 2015 ; Wasmund et al., 2017 ). More comprehensive overviews of the biogeochemical sulfur cycle in marine sediments have been published by, e.g., Canfield (2001) , Amend et al. (2004) , Canfield et al. (2005) , and Jørgensen and Kasten (2006) . Figure 1 presents the sulfur cycle of marine sediments, as it will be discussed in this review. The processes include chemical reactions, microbially catalyzed pathways, and a combination of both. Sulfate (SO 4 2- ) reduction to sulfide (H 2 S + HS - + S 2- ) is driven by the oxidation of buried organic carbon (C org ), supplemented by the anaerobic oxidation of methane (CH 4 ) at the subsurface sulfate-methane transition (SMT). Manganese and iron reduction are focused toward the surface sediment, but Fe(III) is also buried and acts as an oxidant for sulfide in the deeper sediment layers where it partly binds the produced sulfide as iron sulfide (FeS) and pyrite (FeS 2 ). Pyrite is the end product of iron-sulfide mineral formation and provides a deep sink for sulfur. Two pathways of pyrite formation are discussed here, the “polysulfide pathway” (1) and the “H 2 S pathway” (2) ( Figure 1 ). The sulfidization of buried organic matter provides an additional deep sink for sulfur. Intermediate sulfur species, such as elemental sulfur (S 0 ), thiosulfate (S 2 O 3 2- ), tetrathionate (S 4 O 6 2- ), and sulfite (SO 3 2- ), are formed during the oxidation of sulfide by, for example, buried Fe(III). These intermediates may be reduced back to sulfide, oxidized further to sulfate, or disproportionated to form both sulfide and sulfate. In very sulfidic sediments, a part of the sulfide diffuses up to the surface sediment where it may be oxidized by cable bacteria, by large sulfur bacteria such as Beggiatoa spp., or by other, less conspicuous sulfide oxidizers. The different pathways of sulfide oxidation ultimately depend on oxygen (and less on nitrate) as the ultimate oxidant, and thereby consume a considerable part of the total oxygen uptake of the seabed ( Jørgensen, 1982b ). The oxygen flux into the sediment is enhanced by bioirrigation (ventillation of burrows) by the benthic macrofauna (e.g., Kristensen et al., 2013 ). FIGURE 1 The biogeochemical sulfur cycle of marine sediments. The schematic presentation includes many of the processes discussed in this review. Arrows indicate fluxes and pathways of biological or chemical processes. For further explanation, see text."
} | 1,342 |
37255980 | PMC10225898 | pmc | 6,046 | {
"abstract": "The world's population is increasing and is anticipated to spread 10 billion by 2050, and the issue of food security is becoming a global concern. To maintain global food security, it is essential to increase crop productivity under changing climatic conditions. Conventional agricultural practices frequently use artificial/chemical fertilizers to enhance crop productivity, but these have numerous negative effects on the environment and people's health. To address these issues, researchers have been concentrating on substitute crop fertilization methods for many years, and biofertilizers as a crucial part of agricultural practices are quickly gaining popularity all over the globe. Biofertilizers are living formulations made of indigenous plant growth-promoting rhizobacteria (PGPR) which are substantial, environment-friendly, and economical biofertilizers for amassing crop productivity by enhancing plant development either directly or indirectly, and are the renewable source of plant nutrients and sustainable agronomy. The review aims to provide a comprehensive overview of the current knowledge on microbial inoculants as biofertilizers, including their types, mechanisms of action, effects on crop productivity, challenges, and limitations associated with the use of microbial inoculants. In this review, we focused on the application of biofertilizers to agricultural fields in plant growth development by performing several activities like nitrogen fixation, siderophore production, phytohormone production, nutrient solubilization, and facilitating easy uptake by crop plants. Further, we discussed the indirect mechanism of PGPRs, in developing induced system resistance against pest and diseases, and as a biocontrol agent for phytopathogens. This review article presents a brief outline of the ideas and uses of microbial inoculants in improving crop productivity as well as a discussion of the challenges and limitations to use microbial inoculants.",
"conclusion": "4 Conclusion Pesticides and chemical fertilizers are effective for the production and disease control of plants but, their continuous application is a threat to the soil ecosystem, plants as well as human beings. Thus to overcome this problem, use of beneficial microbes as biofertilizers and biocontrol agents is an ecofriendly and cheap method for sustainable agriculture. Biofertilizers have the potential to replace chemical fertilizers as well as pesticides and exert a positive impact on crop productivity and encouragement should be given to its implementation in agriculture. Farmers should be made aware of the benefits of using PGPRs as biofertilizers, and the commercialization of PGPRs should be emphasized. Thus in general we concluded that PGPRs have countless benefits in agriculture. Consequently, we can say that the use of biofertilizers in agricultural fields is the best alternative to chemical fertilizers which influence hazardous effects on flora as well fauna and soil health.",
"introduction": "1 Introduction Microorganisms in the soil play a crucial role in soil biodiversity and coordinated nutrient management. They are essential to the growth and evolution of plants. Recent years have seen the use of chemical fertilizers in agriculture, making the nation more self-sufficient in food production, but at the expense of the ecosystem and the well-being of all living things. The excessive use of these fertilizers in agriculture is expensive and has several negative impacts on soil fertility. To satisfy our agricultural requirements, beneficial microorganisms are better alternatives to conventional farming methods. Biofertilizers are safer than chemical fertilizers because they cause less environmental harm, have more focused activity, and are more efficient when used in lesser amounts. Additionally, they have the capacity to multiply while being concurrently regulated by the plant and local microbes. Additionally, microbial inoculants have quicker decomposition processes and are less likely to cause pathogens and pests to develop resilience [ 1 ]. Bioinoculants do not show any detrimental impact on the soil's plant and animal life as they are ecofriendly, highly efficient, and can be utilized as bio pesticides that do not affect any harmful influence on plant products. The plant requires mineral nutrients which can only be provided when chemical fertilizers are used directly or indirectly, along with organic manure and biofertilizers to increase the organic carbon in soil and uphold sustainability in a field and horticultural crops [ 2 ]. Microbial inoculants are described as organisms that are introduced into an environment for a particular purpose, such as biocontrol or promoting plant growth, such as bacteria, fungi, and other microorganisms [ 3 ]. The term bio-fertilizer refers to a wide range of products that contain living or dormant microorganisms, including bacteria, fungi, actinomycetes, and algae. Upon application, these microorganisms help to fix atmospheric nitrogen or solubilize/mobilize soil nutrients in addition to secreting substances that promote plant growth [ 4 ]. Now a day, biofertilizers and bio pesticides are currently available as substitutes for conventional inorganic fertilizers and synthetic pesticides respectively along with a variety of other products. The market for biofertilizers, which was valued at USD 1.57 billion in 2018, is anticipated to expand at a compound annual growth rate of 12.1% between 2022 and 2027 [ 5 ]. Currently, there are a large number of small and fewer big companies operating across various geographical regions, creating a highly fragmented market. Currently, the market for biofertilizers is dominated by many small companies because it is largely unregulated; however, if regulations are implemented, as has happened in the market for biopesticides globally, it is possible that the market will become more consolidated [ 6 ]. In addition, PGPRs are a special category of microbes that persuade plant defense mechanism and provide resistance to host through the extremely diverse mechanism for further pathogen attack, and considered more beneficial biocontrol agents (BCAs) than typical chemical fertilizers as they are non-pathogenic, naturally inhabitant of the rhizosphere, environment friendly, and enhancing plant yield directly. According to Gupta et al. [ 7 ], PGPRs can influence plant development either directly or indirectly and induce plant growth by deploying mineral nutrients in soils, regulating or inhibiting plants from phytopathogens, generating different plant growth regulators, ameliorating soil structure and bioremediating the soil through separation of noxious heavy metals and lowering chemical compounds such as pesticides, fungicides [ [8] , [9] , [10] ]. Besides, above mentioned functions of PGPRs, it also plays different defense actions in plants by producing antibiotics, siderophores, bio-surfactants and volatiles, and enzymes that vitiate cell wall and brought systemic resistance (ISR). Saharan and Nehra [ 11 ] suggested that a broad range of non-symbiotic and symbiotic bacterial species belonging to the genus Klebsiella, Azotobacter, Azospirillum, Bacillus, Enterobacter, and Serratia were considered as PGPRs. Many researchers are still working on knowing the diversity and significance of biofertilizers and their functions in the improvement of agricultural sustainability. The effects of PGPRs are due to plant age, plant species, soil factors, different stages of growth and various form of soil [ 12 ]. Kumar et al. [ 13 , 14 ] reported that PGPR's role in enhancing nutrient uptake for plants is an essential activity and appropriate for crop development. PGPRs overcome the reduction in plant growth generated by different forms of stresses [ 15 ] including, heavy metals stress [ 16 ], water logging stress [ 17 ], salt stress [ 18 , 19 ] drought stress [ 20 ], and various supplementary hostile environmental situations. PGPRs inoculation soothes plant stress by promoting useful impacts on plant fitness, growth, increasing the production and assimilation of nutrients. Therefore, it is necessary to use PGPRs for ongoing, beneficial agricultural reasons to improve crop yields and soil fertility in challenging conditions. Over the last few decades, PGPRs are increasingly being used for secure and safe agriculture worldwide. The main obstacle to the farmers' success is a lack of high quality bioinoculants available to them. Azotobacter, Azolla, Acetobacter, Trichoderma, Bacillus thuriengensis, and Azospirillum need to receive the proper attention given to them and their use in different cereal and vegetable crops. To boost the soil's organic carbon and keep the sustainability of field and horticultural crops, these biofertilizers should be combined with organic manures and chemical fertilizers [ 2 ]. The schematic representation of different types of PGPRs or microbial inoculants and their significant contribution in crop improvements are documented in Fig. 1 . Fig. 1 Schematic Illustration of PGPRs microbial inoculants. Fig. 1 The aim of this review is to summarize the importance of microbial inoculants used as biofertilizers, and their mechanism to enhance crop productivity. This review highlights a detailed study of direct and indirect mechanisms of bio inoculants, including biological nitrogen fixation (symbiotic and non-symbiotic), phytohormone production, nutrient solubilization (phosphate and potassium), siderophore production etc., and biocontrol of phytopathogen, chitinases, HCN, and others antifungal properties, as biofertilizer to increase crop yield."
} | 2,408 |
39979462 | PMC11842525 | pmc | 6,047 | {
"abstract": "Solitarily foraging ants learn to navigate between important locations by comparing their current view with memorized scenes along a familiar route. As desert ants, in particular, travel between their nest and a food source, they establish stable and visually guided routes that guide them without relying on trail pheromones. We investigated how changes in familiar visual scenes affect the navigation of the red honey ant ( Melophorus bagoti ). In Experiment 1, ants were trained to follow a one-way diamond-shaped path to forage and return home. We manipulated scene familiarity by adding a board on their homebound route just before the nest. In Experiment 2, ants were trained to travel a straight path from their nest to a feeder, and we removed the prominent landmarks on the route after they had established a stable route. We predicted that these scene changes would cause the ants to deviate from their usual straight paths, slow down, scan more, and increase their lateral oscillations to gather additional information. Our findings showed that when the familiar scene was changed, ants oscillated more, slowed their speed, and increased scanning bouts, indicating a shift from exploiting known information to more actively exploring and learning new visual cues. These results suggest that scene familiarity plays a crucial role in ant navigation, and changes in their visual environment lead to distinct behavioral adaptations aimed at learning about the new cues. Supplementary Information The online version contains supplementary material available at 10.1007/s10071-025-01936-3.",
"conclusion": "Conclusion This study has demonstrated that desert ants oscillate as they navigate outbound and homebound. Their paths wiggle and their heads swing left and right. Both these oscillations, along with other behaviors, chief among them stopping to scan, are modulated in the face of scene changes. Scanning increases, the path meanders more, and with a large enough scene change, the amplitude of head swings and the spatial frequency of path oscillations increase. This entire suite of behaviors serves to increase visual exploration and thereby the learning of new visual cues.",
"introduction": "Introduction Ants are known to navigate with a toolkit of path integration, landmark-based guidance, and systematic search (Wehner 2020 ). Path integration involves an insect maintaining a record of the straight-line distance and direction from a starting point, usually its home (Wehner and Srinivasan 2003 ; Wehner and Wehner 1986 ). The vector computed by path integration is now believed to be represented within the central complex of the insect brain, specifically in a neural structure that, while not anatomically arranged in a ring, operates as a ring attractor network (Heinze et al. 2018 ; Lyu et al. 2022 ). The bump of activity on each part of the neural structure indicates a direction and its amplitude codes distance. In view-based navigation, the ant compares its current view with remembered views at the goal and along the route to the goal (Cheng 2012 ; Le Moel and Wystrach 2020 ; Wehner 2003 ; Wehner and Räber 1979 ). In using views, ants are now thought to learn scenes to head toward (attraction) and scenes to turn away from (repulsion, Le Moel and Wystrach 2020 ; Murray et al. 2020 ). In systematic search, which is frequently needed near the nest when the former two strategies do not take the ant exactly to the nest entrance, the navigator travels in loops that increase in size as the search goes on (Schultheiss et al. 2015 ; Wehner and Srinivasan 1981 ). Loops also feature prominently in learning views. In a number of species that have been studied, a would-be forager first takes loops around its nest before heading off to forage in what are called learning walks , three to seven such walks in bull ants ( Myrmecia croslandi , Jayatilaka et al. 2018 ) and desert ants ( Cataglyphis bicolor : Wehner et al. 2004 ; C. noda and C. fortis : Fleischmann et al. 2016 ; Fleischmann et al. 2018 ; Melophorus bagoti : Deeti et al. 2020 ; Deeti and Cheng 2021a ; Deeti et al. 2024a ; reviews: Freas et al. 2019 ; Zeil and Fleischmann 2019 ). During learning walks, the view learner often stops and looks in various directions, turning on the spot in what are called scanning bouts (Deeti et al. 2023a ) or pirouettes (Fleischmann et al. 2017 ), presumably to learn what the scene looks like in various directions, including the direction to the nest. Ants also scan in bouts on some trips home, especially when experimenters have changed the scene through some manipulation (Deeti et al. 2023a ; Wystrach et al. 2014 ). Presumably, such scans help a navigator to find the best direction to home in. Even on a familiar route, with or without scanning at the start of the trip, ants do not strike a straight path home. Rather, they oscillate laterally, if only slightly, with the path ‘meandering’ left and right as the ant travels (Clement et al. 2023 ). Ants, bull ants M. croslandi and meat ants Iridomyrmex purpureus in the study, oscillate more when the visual scene is unfamiliar than when the scene is familiar. Oscillatory behaviors are crucial for navigation and widespread across life, from bacteria to animals (Cheng 2022 , 2023 , 2024 ). The system or mechanism responsible for oscillatory behavior is termed an oscillator , said to be a basic unit of action (Gallistel 1980 ), but perhaps is a basic unit of life (Cheng 2022 , 2023 ). Oscillations of effectors that drive movement are common to mobile organisms, but lateral oscillations add a new dimension: the traveler can compare conditions on the two sides as it winds left and right and gather more information about the world as it moves. The lateral oscillatory behaviors of ants in navigation have been described in the past decade (Clement et al. 2023 ; Collett et al. 2014 ; Dauzeres-Perez and Wystrach 2024 ; Lent et al. 2013 ; Murray et al. 2020 ). What we want to add to this topic are measures of the characteristics associated with oscillations, frequency, amplitude, and in one case, phase relations between two different oscillations. We aim to build upon the extant work on lateral oscillations in ants by focusing on one of the extensively studied species of desert ants, the Australian red honey ant Melophorus bagoti . We measured the number of bouts of scanning and two kinds of oscillations evident in the videos of moving ants: lateral path oscillations and oscillations or swings of the head from side to side. We defined and measured the frequency and amplitude of these oscillations. For path oscillations, we measured frequencies in time and space. We also examined the (temporal) phase relationship between these two kinds of oscillations, that is, where the head-swing cycles fall on the lateral path oscillations. In addition, we calculated three different measures of straightness to characterize the overall extent of oscillations. We hypothesized, based on findings from studies on meat ants and bull ants (Clement et al. 2023 ), that in visually unfamiliar circumstances, oscillations would increase. In Experiment 1, we forced ants to travel one-way around a diamond to forage for food and return home. We videotaped the ants from above on their last leg before reaching home. To manipulate scene familiarity, we added a piece of board placed on the side of the homebound track right before the ants reached home. In Experiment 2, we placed prominent boards enroute a straight path from the ants’ nest to a feeder and removed the landmark set up after ants were trained to travel the outbound route. We predicted that the paths with a scene change would be less straight on all straightness measures, that the ants would slow down, that they would scan more, and oscillate more. Our suite of measures of oscillations will let us know in which ways the ants oscillate more. Slowing down, scanning more, and oscillating their heads and their paths more would all serve to gather more information rather than simply exploiting known information to get home as quickly as possible, a theme that we discuss later.",
"discussion": "Discussion Our experimental view changes did not affect navigational success in the desert ants, as all of them arrived successfully at their goals. But the addition or especially the removal of boards along the route changed the walking characteristics and oscillations of the ants. The ants slowed down, scanned more and for longer durations, and their paths were less straight. The ants engaged in at least three types of oscillations: the legs lifted and planted in cycles, the head swung left and right, and the path wiggled left and right. With view changes, the head swung more to the left and right, increasing amplitudes of orientation oscillations. With board removal, although not with board addition, spatial frequency of path oscillations increased. These results demonstrate the existence of oscillators at work in navigation (Clement et al. 2023 ), with navigational demands adjusting the working of oscillators (Cheng 2022 , 2023 ). The ants’ reactions to view changes and the workings of oscillators form main themes for this discussion. Reactions to view changes The suite of behavioral changes in navigation in the face of a scene change makes sense as strategies to gather more visual information about the environment. An ant traveling with slower speed than usual, with more meander, more turning of the head, and more stopping to scan for longer would be looking at the environment in more directions and from more places than usual. With a scene change, the ants searched and likely learned the new information. As ants showed oscillations, especially head turns, even on the paths well traveled (in the Control conditions), they were engaging in some information seeking in all navigation. The balance between exploration, the seeking of potentially new information, and exploitation, striding off the well-known route (Clement et al. 2023 ), tips to more exploration with view changes. But the trade-off between exploitation and exploration, first noted to our knowledge by Kramer and Weary ( 1991 ), is still in play in routine runs home or in ‘control’ conditions, testifying to the importance of exploration (Brembs 2011 , 2021 ). The ‘imperfections’ or variability in the oscillatory cycles inject variability in behavior, an important component in exploring. With regard to other species, view changes on a well-traveled route reduced night-active bull ants’ navigational efficiency significantly (Islam et al. 2020 , 2021 ; Narendra and Ramirez-Esquivel 2017 ). While the view changes observed in Islam et al.’s ( 2020 , 2021 ) studies were large, the view change in Narendra and Ramirez-Esquivel’s ( 2017 ) study was subtle, consisting in the felling of three trees in a forested surround. The authors used arrows in their illustrative figure to point out the change caused by the tree felling; the difference was not easy for readers to spot. The red honey ants, in contrast, cope with view changes with aplomb, continuing to navigate successfully after being raised a meter off the ground (Julle-Daniere et al. 2014 ) or with one side of the usual view blocked (Schwarz et al. 2014 ). None of these studies examined oscillatory characteristics. The bases of these differences in reactions are unclear. Species differences and night vs. day travel both come to mind as possibilities. Our study found that board removal on the outbound route had larger effects than board addition on the inbound route. Although this is not a focus of the current study, we believe that the difference stems from the much larger view change in the board-removal manipulations. In Experiment 2, the boards on the outbound route were a good deal larger than that in Experiment 1, and two of them stood right on the path to the goal (feeder). Future studies might examine ‘dose–response’ curves in reactions to visual changes. Previous studies on desert ants have found an increase in scanning or meandering or both when matters are not usual in travel (Freas et al. 2022 ; Wystrach et al. 2014 , 2019 , 2020 ). When views are unusual at the start of the journey home, ants scan more (Wystrach et al. 2014 ). When an ant had been picked up near the end of its journey home and placed back on the route, the traveler still heads in the home direction, but scans and meanders more (Wystrach et al. 2019 ; in bull ants, Deeti et al. 2023c ; Lionetti et al. 2024 ). When an ant had encountered a trap that delayed its trip home, it scans more as it nears the location of the trap on the subsequent trips (Freas et al. 2022 ; Wystrach et al. 2020 ). We theorize that all these manipulations induce something equivalent to uncertainty in the ants. Whatever proxy measures for uncertainty the ants are relying on, the outcome can be summarized as more exploration under conditions we experimenters would call uncertain, again reflecting the trade-off between exploration and exploitation (Brembs 2011 , 2021 ; Clement et al. 2023 ). Finally on this topic, we have replicated the pattern of reactions to view changes in a separate study on Nest 2 for a different purpose (Deeti et al. 2024e ). Changing the color of the boards on the way to the feeder also led to increased scanning and meander, higher spatial frequency of path oscillations, and higher amplitude of head oscillations. Oscillators and oscillations We found path oscillations and head oscillations, the former replicating similar findings in day-active Australian bull ants ( Myrmecia croslandi ) and meat ants ( Iridomyrmex purpureus ; Clement et al. 2023 ), in wood ants ( Formica rufa ; Collett et al. 2014 ; Lent et al. 2013 ), and in desert ants Cataglyphis velox (Dauzeres-Perez and Wystrach 2024 ). Path oscillations may thus be common in ants, and it is also found in many other insects (Clement et al. 2023 ; Namiki and Kanzaki 2016 ). Whereas Clement et al. ( 2023 ) used sophisticated Fourier analysis to extract characteristics of oscillations, our approach was to base calculations on the x–y coordinates extracted from the video records. Aside from some smoothing, also used in the Clement et al. ( 2023 ) study, our definitions of frequency and amplitude used the spreadsheet data without further recoding or modeling. That such measures can be readily measured based on x–y coordinates of the locations of chosen points on the ants’ body testifies to the robustness of head and path oscillations in ant navigation. Navigational servomechanisms operate with intrinsic oscillators in animals and even non-neural organisms (Cheng 2022 , 2023 ), with the oscillators responsible for generating periodic movements of effectors that drive locomotion known as oscillations. Oscillators come with costs. Compared with random-rate processes in behavior, which might rely on noise in the systems generating behavior (Berg and Brown 1972 ; Deeti et al. 2023a , 2024f ; Scharf et al. 1998 ), a dedicated mechanism is needed to orchestrate oscillations, presumably relying on neurons in animals. With head and path oscillations seemingly independent in our data set, two such systems are needed. Both neurons and wiring come with hefty costs (Sterling and Laughlin 2015 ). An additional cost is the time taken to weave left and right, compared with a straight course. These basic units of action (Gallistel 1980 )—oscillators generating lateral oscillations—so common in insects (Clement et al. 2023 ; Namiki and Kanzaki 2016 ) and other organisms (Cheng 2022 , 2023 ) must provide benefits. Our discussion of reactions to visual changes has already identified a key benefit: exploration. Frequency and amplitude can be considered as ‘free’ parameters that can be adjusted servomechanistically to serve navigational functions. Variability in behavior in general has other benefits than exploration, including evading predators and searching a ‘problem space’ for optimal solutions to a behavioral challenge (Brembs 2011 , 2021 ). Oscillations have yet another benefit in the normal, familiar course of travel without any scene change: course control (Wystrach et al. 2020 preprint). Adjustments, perhaps only small ones, to the left and right keep the traveler on the best course to head to its goal. The basic operation of the view-based navigational servomechanism in ants may be to command a turn to the left or turn to the right. Oscillating left and right keeps the ant on course in heading toward the ‘best’ view. If one keeps to a ruler-straight course of travel, one soon becomes uncertain as to whether the travel direction is still the best direction, there being a lack of comparisons with views in other directions (Cheng 2023 ). Managing uncertainty might be another function of oscillations."
} | 4,220 |
36300582 | PMC10099496 | pmc | 6,049 | {
"abstract": "Abstract The spatial organization of biofilm bacterial communities can be influenced by several factors, including growth conditions and challenge with antimicrobials. Differential survival of clusters of cells within biofilms has been observed. In this work, we present a variety of methods to identify, quantify and statistically analyse clusters of live cells from images of two Salmonella strains with differential biofilm forming capacity exposed to three oxidizing biocides. With a support vector machine approach, we showed spatial separation between the two strains, and, using statistical testing and high‐performance computing (HPC), we determined conditions which possess an inherent cluster structure. Our results indicate that there is a relationship between biocide potency and inherent biofilm formation capacity with the tendency to select for spatial clusters of survivors. There was no relationship between positions of clusters of live or dead cells within stressed biofilms. This work identifies an approach to robustly quantify clusters of physiologically distinct cells within biofilms and suggests work to understand how clusters form and survive is needed. Significance statement Control of biofilm growth remains a major challenge and there is considerable uncertainty about how bacteria respond to disinfection within a biofilm and how clustering of cells impacts survival. We have developed a methodological approach to identify and statistically analyse clusters of surviving cells in biofilms after biocide challenge. This approach can be used to understand bacterial behaviour within biofilms under stress and is widely applicable.",
"conclusion": "CONCLUSIONS This work demonstrates in controlled conditions the differences in efficacy between three common oxidizing biocides against biofilms of 2 Salmonella strains and established that bacterial survival is strain, exposure time and biocide dependent. This shows that designing effective biocidal regimes requires data from diverse strains to ensure adequate coverage of potentially more tolerant strains when in a biofilm context. Improving our understanding of differences in how strains respond to biocidal challenges shows that testing biocide regimes should include use of strains with different biofilm formation capacities as well as different biocide concentrations to predict efficacy. One of the most popular supervised learning methods, SVM with RBF kernel, has been applied for binary classification of strains as weak or strong biofilm formers. We conducted 10‐fold and leave‐out‐one cross‐validations to test the model, and performed SVM assessment, by evaluating sensitivity and specificity. We note that increasing sensitivity and specificity lowers the probability of type II and type I error, respectively. We expect other algorithms to perform similarly and although we did not show the results, we also tried a random forest which resulted in a similar performance. (With only 18 data points, we do not anticipate classification algorithms to vary a lot in performance.) Given 3D confocal microscopy data of thick biofilms and enough samples for given biofilm‐forming strains or treatments, a PCA‐SVM classifier might also be used to extend the analysis. A similar technique involving different antibiotics has been applied in Yoram et al. (2018). Most statistical approaches addressing the spatial distribution of biofilm cells rely on spatial analysis methods such as Ripley's K , and to a lesser extent on methods of spatial randomness such as Hopkins statistic. In addition to these two methods, we applied multimodality tests (Classic Dip test and Classic Silverman test) and related tests on reduced versions of the data (PCA Dip and Dip‐dist), supported by clusterability fraction, to evaluate clusterability in both strains. Although computationally expensive, the advantage of our approach is to calculate the clusterability fraction , that could be compared across different conditions, at the same level of significance. We hope to extend this approach to coupling data describing particle counts and certain chemical entities (e.g., metabolites, autoinducers), obtained by confocal microscopy. Survival within biocide treated biofilms was not uniform which supports previous work suggesting large numbers of cells are sacrificed in a process of impeding biocide penetration (Diez‐Garcia, 2012 ). However, clustering of survivors was seen even in relatively immature biofilms (e.g., SL1344 at 24 h) which makes differential susceptibility between clusters more likely than a physical protective effect. The clustering observed is likely to reflect differences between clusters that are imprinted as the initial seeding of microcolonies by individual cells. Differences in genotype, gene expression or other epigenetic features which vary between clusters are probably related to likelihood of survival. This study demonstrates clear differential behaviour of clusters of cells in terms of survival within biofilms as well as outlining a framework to identify and quantify clusters of live or dead cells. The mechanistic basis which dictates differential survival of these clusters is uncertain and future work to study gene content, expression and behaviour of single cells within clusters is needed. Understanding how clusters establish, differentiate from each other and survive stress will help develop strategies to control biofilm formation and is an important future goal.",
"introduction": "INTRODUCTION Biocides are crucial for control of microbial contamination and infection and are used in a wide range of clinical, industrial, veterinary, and domestic settings (Linley et al., 2012 ). Whilst many common biocidal agents have been employed for decades there are still major gaps in our understanding of mechanisms of action and resistance. Oxidizing biocides have a broad spectrum of activity, similar chemistries, and proposed mechanisms of action. The basic mechanism by which they exert their biocidal activity is thought to be via damage to cellular macromolecules (Finnegan et al., 2010 ). They are usually low molecular weight compounds able to enter the bacterial cell, thereby accessing intracellular targets, although there is also evidence for antimicrobial action exerted at the cell wall and membrane for some compounds (Finnegan et al., 2010 ). The three most used common oxidizing biocides are hydrogen peroxide, peracetic acid and sodium hypochlorite. Hydrogen peroxide (H 2 O 2 ) is widely used for disinfection, sterilization and antisepsis and degrades into non‐toxic by‐products of water and oxygen making it an attractive choice for any application involving food production (Rutala & Weber, 1999 ). The biocidal activity of hydrogen peroxide may be due to interactions with intracellular iron forming iron ions and hydroxyl radicals (Finnegan et al., 2010 ) formed by the Fenton reaction. Several studies have shown hydrogen peroxide to be responsible for damage to DNA (Henle & Linn, 1997 ) proteins (Imlay et al., 1998 ), amino acids (Dean et al., 1997 ), and cell membranes (Baatout et al., 2006 ; Brandi et al., 1991 ; Peterson et al., 1995 ). Peracetic acid (PAA) is a peroxide of acetic acid (Block, 1991 ). It is soluble in water and exists in equilibrium between acetic acid (CH 3 CO 2 H), peracetic acid (CH 3 CO 3 H), water (H 2 O) and hydrogen peroxide (H 2 O 2 ). It is a weak acid with a p K \n a of 8.2 (Unis, 2010 ). It has greater oxidizing potential than chlorine or chlorine dioxide and is environmentally safe due to its degradation into non‐toxic components (Kitis, 2003 ). It also has higher antimicrobial activity than hydrogen peroxide (Wagner et al., 2002 ) and remains active in the presence of interfering matter including organic material such as blood (Russell & McDonnell, 1999 ). The mechanism of action of peracetic acid has not been fully investigated but it has been hypothesised that it disrupts sulphydryl (—SH) and sulphur (S—S) bonds in biomolecules (Russell & McDonnell, 1999 ). It has also been suggested that PAA disrupts the cell wall and cell membrane by oxidizing structural lipoproteins and when acting intracellularly may inactivate vital metabolic enzymes and DNA bases (Kitis, 2003 ). Chlorine was first discovered by Scheele in 1774 (Rutala & Weber, 1997 ) and has a wide range of antimicrobial activity. Sodium hypochlorite (NaClO) is a salt of the hypochlorite ion dissolved in water which in solution can dissociate to give hypochlorous acid. Hypochlorous acid has considerably more antimicrobial activity than the hypochlorite ion and is responsible for much of the antibacterial efficacy (Rossi‐Fedele et al., 2011 ). Sodium hypochlorite has a broad spectrum of activity with its primary industrial use being water treatment (Rutala & Weber, 1997 ). Proteins, peptides, lipids and DNA have all been shown to be oxidized by sodium hypochlorite at physiological pH with C=C double bonds, peptide bonds, peptide groups and thiol groups susceptible to electrophilic damage (Fukuzaki, 2006 ). Notwithstanding this reactivity, it is postulated that the primary action of the biocide is oxidative damage to DNA synthesis since low concentrations of sodium hypochlorite leave protein synthesis far less affected than DNA synthesis (Russell & McDonnell, 1999 ). Concerns have been raised about the possibility of biocide resistance emerging. It is generally not possible for bacteria to achieve resistance to in use biocide concentrations, but studies have documented strains able to survive low levels of biocide (tolerance) and identified mutants isolated after biocide exposure with cross resistance to antibiotics (Copitch et al., 2010 ; Karatzas et al., 2008 ; Randall et al., 2007 ; Walsh et al., 2003 ). Bacterial biofilms are far more resistant to biocide challenge than planktonic cells (Bansal et al., 2019 ; Russo et al., 2013 ; Vestby et al., 2009 ). The reasons for these significant changes in bacterial susceptibility to biocides have been proposed to relate to the structure of the bacterial biofilm and the EPS matrix, altered metabolism in the biofilm phenotype, persister cell generation and an increase in genetic transfer. Biofilms as a community of cells encounter a range of internal and external stresses resulting from nutrient limitation, waste product secretion, ecological competition, desiccation, and antimicrobial exposure (Hall‐Stoodley et al., 2004 ). Biofilms are inherently resilient to stress (Rode et al., 2020 ) in part due to the heterogeneity of cells and structures within the community, cells can be spatially organized into clusters and this has been linked to antimicrobial survival (Wong et al., 2021 ). To characterize the spatial distribution of cells, digital image analysis (Dazzo & Yanni, 2017 ; Schillinger et al., 2012 ), spatial analysis methods such as Ripley's K (Hart et al., 2019 ; Ishkov et al., 2021 ; Marchal et al., 2017 ) and methods for determining spatial randomness such as the Hopkins statistic (Drury et al., 1993 ; Espinoza et al., 2012 ) have been used. However, methods for evaluating clusterability can vary significantly and a comprehensive comparison of statistical tests in the biofilm context is currently lacking. The aim of this study was to employ a combination of microscopy and statistical approaches to identify cell survival and the impact of spatial distribution of cells within a biofilm on biocide susceptibility. We investigate the effect of three oxidizing biocides against two Salmonella enterica subsp. enterica strains (one serovar Typhimurium and one Agona) with different biofilm forming capacities. First, with a support vector machine approach, we showed spatial separation between the two strains. Second, we applied five statistical tests, four of which are new in the biofilm setting, and used HPC, to evaluate clusterability. Our results indicate that biocides of medium potency, like NaClO, have a stronger tendency to select for spatial clusters of surviving cells in S . Typhimurium biofilms. Finally, we showed that simple visualizations of confocal images can efficiently quantify colocalization between live and dead cells and that there is no link between the spatial locations of live and dead clusters of cells within stressed biofilms.",
"discussion": "RESULTS AND DISCUSSION Weak and strong biofilm forming strains can be distinguished by support vector machine‐based differentiation using confocal microscopy images We used confocal microscopy images to identify live and dead cells (based on staining with SYTO‐9 and propidium iodide) for two Salmonella strains with different biofilm forming capacities ( S . Typhimurium SL1344, a relatively modest biofilm former, and S . Agona 3750, a strong biofilm forming strain, Figure S1 ). We then aimed to compare the ability of each to survive exposure to oxidative biocides. Our first goal was to quantify differences in biofilm formation from image data. For each image, the number of particles (cells) were extracted, added unity and log10‐transformed. Mean and standard error (SE) of flow cell coverage for both strains at 24 and 48 h are shown in Figure 2 and confirmed the expected greater increase in biomass in strain 3750 versus SL1344 (representative images and average flow cell coverage are shown in Figure S1 ). FIGURE 2 Mean and SE of the number of particles for 19 different conditions (Table S1 ). Red and black colours show discrimination between weak (SL1344) and strong (3750) biofilm forming strains, respectively. To assess whether the underlying 1D probability distributions between the two strains differ, we performed two‐dimensional two‐sample Kolmogorov–Smirnov (KS) test, a generalization of the classical KS test, using the transformed data with coordinates denoting the mean number of particles at 24 h and 48 h (Fasano & Franceschini, 1987 ). The difference between the mean number of particles in the 2D space (24 h, 48 h) from strains SL1344 and 3750 was statistically significant. p ‐value < 0.01). Support vector machines (SVM) are learning algorithms which aim to identify a function (hyperplane) that can separate datasets. Biomedical applications of SVM include the classification of bacterial species to distinguish between disease conditions (Yoram et al., 2018 ) or the innate fluorescence signatures of microbial cells (Yawata et al., 2019 ), prediction of biofilm‐inhibiting‐peptides (Gupta et al., 2016 ), classification of antibiotics (Jung et al., 2014 ) or differentiation of human and in vitro biofilm transcriptomes (Cornforth et al., 2018 ). Here we applied an SVM classifier with radial basis function (RBF) kernel to differentiate between the weak (SL1344) and strong (3750) biofilm forming strains using the mean number of particles from a single 2D plane of Z‐stack for each condition. The data were split into 58% training (11 data points) and 42% test (8 data points). We estimated γ, the RBF kernel parameter, directly from data points in the training set X ⊂ R 11 × 2 , using the formula in Li et al. ( 2011 ) \n γ = 1 τ 2 , τ = 1 11 2 ∑ i < j , j = 2 11 X i − X j 2 , \n where ||·|| is the Euclidean distance and τ \n 2 is an estimate of the variance in the data and X i is the i th observation used in the sample, consisting of the mean number of particles at 24 and 48 h in a given Z‐stack. Then, for a fixed γ = 0.27, 10‐fold cross‐validation was performed to select the best misclassification cost, C , using the tune function from the e1071 library ( https://cran.r-project.org/web/packages/e1071/index.html ). For the optimal C = 12%, 35% of test observations are subject to misclassification. The level of prediction accuracy on the test data is shown by calculating the receiver operating characteristics (ROC) curve in Figure S2 . The SVM algorithm tries to create a decision boundary such that the separation between two classes of data is as wide as possible. The misclassified points and those closest to this hyperplane are the support vectors. These are shown in Figure 3A as well as the fitted decision boundary that was generated by the RBF kernel by learning from data. The data show the SVM has generated a clear separation between the green and blue data points corresponding to the SL1344 and 3750 biofilms respectively, further confirming their inherent difference in biofilm formation. FIGURE 3 Decision boundary generated by the SVM classifier and support vectors in diamonds. The mean number of particles could distinguish between weak and strong biofilm forming strains with relatively high accuracy. γ = 0.27, C = 12 in a, and γ = 0.8, C = 5 in B. We also performed 10‐fold cross‐validation for both parameters γ and C . For γ = 0.8, C = 5, the decision boundary correctly classified all training points. Although only 15% of test observations are subject to misclassification and Figure 3B is seemingly more accurate than Figure 3A , these results are less robust. This is because several runs of 10‐fold cross‐validation are required for correct binary classification. To evaluate the performance of classification algorithms for Figure 3A , we used leave‐out‐one cross‐validation (LOO‐CV), when a partition is made up of 18 training data and 1 testing. This is a special case of K ‐fold validation, when K equals the number of data points in the set. The parameters γ and cost of constraints violation were estimated using the same approach as above 19 separate times. Except 2, all data points were correctly classified by LOO‐CV, thus predicting a high (89.5%) classification accuracy. Our results show that the SVM approach could accurately distinguish between strains with different biofilm forming capacities using confocal image data at 24 and 48 h, regardless of treatment. Microbial colonies within Salmonella biofilms are non‐randomly distributed Non‐random spatial organization of cells and matrix components are believed to contribute to the persistence of biofilm communities as these structures provide structurally distinct microenvironments as well as promoting physiological heterogeneity of cells within a community (Petersen et al., 2019 ). In support of this, a non‐uniform distribution of surviving cells within Salmonella biofilms after biocide challenge was observed by confocal microscopy (Figure S3 ) and this differed between test biocides. We used both spatial randomness tests (Hopkins statistic), multimodality tests (Classic Dip test and Classic Silverman test) and related tests on reduced versions of the data (PCA Dip and Dip‐dist) to study and evaluate the clusterability of survivor positions in the different conditions. These tests were applied to the 2D plane of Z‐stack. Hopkins statistic tests for spatial randomness and evaluates if any feature is distributed non‐randomly across the data set (Hopkins & Skellam, 1954 ; Lawson, 1990 ). Although Hopkins may be preferred when small clusters are of interest (Adolfsson et al., 2017 ), it has only been used in a few biofilm‐ or receptor aggregate‐related studies. Examples are studies on the interaction of latex particles with P. aeruginosa (Drury et al., 1993 ) and the quantification of clustering of membrane proteins labelled with gold nanoparticles (Espinoza et al., 2012 ). The ‘hopkins’ function from the clusterend R package (Adolfsson et al., 2017 ) was used to calculate the Hopkins statistic for each of our conditions, which distinguishes non‐clustered from moderately or highly clustered distributions. The Classic Dip test rejects the null hypothesis of unimodality if the empirical distribution is sufficiently different from the closest possible uniform one (Hartigan & Hartigan, 1985 ). The Classic Silverman test is based on the kernel density estimate and it will reject the assumption of unimodality if a mixture of distinct Gaussian distributions is required to produce the underlying empirical distribution (Silverman, 1981 ). The ‘modetest’ function from the multimode R package ( https://cran.r-project.org/web/packages/multimode/multimode.pdf ) and the ‘dip.test’ from the diptest R package ( https://cran.r-project.org/web/packages/diptest/diptest.pdf ) were used for Silverman's mode estimation method and Hartigan's dip statistic. PCA Dip (Dip test on principal components) and Dip‐dist (Dip test on pairwise distances) uses the classic Dip test to test whether the first principal component is multimodal (Adolfsson et al., 2017 ) or to test for clusters on a set of pairwise distances (Kalogeratos & Likas, 2012 ). The codes for PCA Dip and Dip‐dist were implemented using the diptest R package by using the first principal component and the matrix of pairwise distances. We ran all five methods with default parameters in a series of simulations to evaluate the clusterability of data using HPC. For the multimodality tests 1000 runs for the Classic Dip test, PCA Dip, Dip‐dist and 100 runs for the Classic Silverman test were performed. The percentage of data sets on which the tests yielded a p ‐value less than 0.05 was recorded, indicating that the test rejected the assumption of unimodality at 5% significance level. For unambiguously unclusterable image datasets, the proportion of rejections corresponds to a Type I error (the rate of incorrectly classifying unclusterable data sets as clusterable) (Adolfsson et al., 2017 ). For the Hopkins statistic, clusterability can be inferred from a threshold based on the Beta distribution. Under a null hypothesis, the test statistic H will follow a Beta distribution with both parameters equal to n, the number of data points sampled (Adolfsson et al., 2017 ; Hopkins & Skellam, 1954 ; Lawson, 1990 ). Hence, the Beta statistic should be compared to the Beta quantile qα ( n , n ) α , which is defined as the probability of concluding that the data is clustered, assuming it was generated without clusters, that is, p ( H < qα ( n , n )) is 100 α %. The recommended sampling rate for n is 5%–10% of the data (Lawson, 1990 ). We randomly sampled 10% of the data in a series of 1000 runs and following (Adolfsson et al., 2017 ), we used a one‐sided test with α = 0.05. Each of these methods can capture the structure of the data differently, to take a consistent approach, we considered data from a given experimental condition clusterable if a replicate has at least 25 particles and the clusterability fraction exceeds 80% in at least two biological replicates of the same condition by at least one of the five tests. It is defined as the proportion of p ‐values less than 0.05 out of all runs. The summary of conditions where cluster structure was detected by this criterion is in Table 1 . The (1)–(3) denote the replicates of a given condition. TABLE 1 Conditions where clustering of survival cells was identified by statistical tests Hopkins's test Classic dip Classic Silverman PCA dip Dip‐dist 1000 runs 1000 runs 100 runs 1000 runs 1000 runs Images \n p < 0.05 \n p < 0.05 \n p < 0.05 \n p < 0.05 \n p < 0.05 Number of particles SL1344 20 min H 2 O 2 48 h (1) 100% 1521 SL1344 20 min H 2 O 2 48 h (2) 100% 1425 SL1344 20 min H 2 O 2 48 h (3) 100% 1515 SL1344 20 min NaClO 24 h (2) 100% 100% 98 SL1344 20 min NaClO 24 h (3) 100% 100% 162 SL1344 20 min NaClO 48 h (1) 99% 650 SL1344 20 min NaClO 48 h (2) 83% 591 SL1344 40 min NaClO 48 h (1) 100% 100% 100% 81.2% 751 SL1344 40 min NaClO 48 h (2) 100% 832 SL1344 40 min NaClO 48 h (3) 100% 100% 100% 685 SL1344 40 min PAA 24 h (1) 100% 28 SL1344 40 min PAA 24 h (2) 100% 100% 35 SL1344 40 min PAA 24 h (3) 100% 35 SL1344 40 min H 2 O 2 48 h (1) 97.8% 100% 95% 96 \n SL1344 40 min H 2 O 2 48 h (2) \n 100% 421 SL1344 40 min H 2 O 2 48 h (3) 100% 401 SL1344 48 h control (1) 99% 1307 SL1344 48 h control (2) 100% 1195 SL1344 60 min NaClO 48 h (1) 95.6% 100% 100% 100% 582 SL1344 60 min NaClO 48 h (3) 100% 382 3750 20 min PAA 24 h (1) 100% 47 3750 20 min PAA 24 h (3) 100% 100% 25 \n Note : The percentage represents the clusterability fraction, that is, the proportion of p ‐values less than 0.05 out of all runs. Altogether 8 out of the 19 conditions possessed a sufficient cluster structure and were meaningfully partitioned in at least one of the time points (24 h or 48 h). In Table 1 , replicates without clustering structure were omitted, thus giving a total of 22 images across 8 conditions. All five tests had a very high clusterability fraction (>95%) except Classic Silverman and Dip‐dist for the SL1344 20 min NaClO 48 h (2) and SL1344 40 min NaClO 48 h (1) samples. The Classic Dip and Silverman tests showed differences in clustering between conditions, for example, SL1344 20 min NaClO 24 h (2–3) samples have a relatively small number of sparsely distributed particles, and thus, methods that account for outlier robustness like Dip test, may be more effective. In contrast, SL1344 20 min H 2 O 2 48 h (1–3), SL1344 20 min NaClO 48 h (1–2) and SL1344 48 h control (1 and 3) samples had many uniformly distributed particles and as such, methods that allow for small clusters like Silverman test may be more appropriate (Adolfsson et al., 2017 ). The Hopkins statistic performed consistently with the Classic Dip test, except for the 3750 20 min PAA 24 h condition. The PCA Dip and Dip‐dist methods had a high clusterability fraction (>80%) for at least one sample across four conditions (SL1344 20 min NaClO 24 and 48 h counted once). Although PCA Dip and Dip‐dist are mostly recommended for clustering of high‐dimensional data sets (Adolfsson et al., 2017 ), they were able to detect a clustering structure in samples where the Classic Dip was not. We conclude that classic multimodality tests complemented with their counterparts on reduced versions of the data, might be preferred to analyse the spatial distribution of live cells. We assessed the degree of clusterability in each condition by the number of times high clusterability fraction (>80%) was detected across replicates by the 5 statistical tests in Table 1 (Figure 4 ). FIGURE 4 The relative degree of clustering observed in the eight conditions. This shows clustering was most likely in biofilms formed by SL1344 (weak) compared to 3750 (strong) Clustering of survivors under stress is not random Previous studies have shown a high level of bacterial survival in biofilms treated with NaClO and H 2 O 2 (Flach et al., 2016 ; Lin et al., 2011 ). This was also the case here (Figure S4 ) and we identified a non‐uniform distribution of live cells by analysing confocal microscopy images. Figure 4 shows that SL1344 biofilms treated with biocides of medium to low potency (NaClO, H 2 O 2 ) for 20–40 min possess an inherently clustered structure. Changing the length of dosing from 20 to 40 min affected clustering significantly for NaClO‐treated strain SL1344 biofilms at 48 h. The mean number of particles in an image was smaller for the 20 min than for the 40 min NaClO treatment (675.3 and 756, respectively). This is not surprising, given the reduced duration of exposure time to NaClO. The statistical tests also revealed that there is clustering of live cells present in untreated SL1344 biofilms at 48 h which becomes more marked. This phenomenon has been observed before in other single‐species biofilms (Kara et al., 2007 ) and is likely to reflect the underlying structure resulting from cells embedded in an extracellular polysaccharide matrix (EPS), which can stimulate cluster formation. There was however a noticeable lack of clustering detected in 3750 biofilms except the 3750 20 min PAA 24 h condition, (which had a relatively low bacterial survival due to the high potency of PAA). In our system, time had a large impact on the clusterability of image data sets from 3750 biofilms exposed to biocides of medium to low potency. This may reflect the decrease in biocide efficacy against the more robust biofilms formed by 3750 for 48 h compared to 24 h, resulting in a low level of selection for survivors which would be consistent with some other studies (Stewart, 2015 ). We postulate that the formation of cell clusters provides protection against less potent oxidative biocides and that this becomes more pronounced as exposure is prolonged. Clustering was also more obvious in weaker biofilm forming strains where fewer cells are surviving. The transient (24 h) and more mature (48 h) cell clusters are depicted in Figure 5 (using the same data as depicted in Figure 2 ). FIGURE 5 Mean and SE of the number of particles for 19 different conditions. Colours denote cell clusters at 24 h or 48 h. cluster structure is present in 72.7% of the conditions associated with strain SL1344 in at least one of the time points. Squares around the data points represent 3750 biofilms and points with no squares represent SL1344 biofilms Given the identical treatment conditions between the two strains, one unexpected result in Figure 5 is that at 24 h strain SL1344 biofilms appear to survive biocide exposure better than strain 3750. This is an anomalous result since the opposite would be expected given the relatively weaker biofilm formation of this strain (Figure 2 and S1 ). This transient growth advantage might be linked to metabolic switching observed in Martins et al. ( 2013 ) and consequently, increased growth and cell aggregation to withstand concentrations of oxidizing biocides. Analysis of single images from a biofilm can be complicated by blurring as they are taken as slices through a three‐dimensional structure, 3D or 2D visualization of clusters can help resolve this and help analysis of whether clusters are spaced randomly. Figure 6A shows the green channel (live cells) of SL1344 grown for 48 h and exposed to NaClO for 40 min. Figure 6B shows the 3D histogram of the Z ‐stack image, the number of bins was chosen such that the maximum number of particles in a bin is 13, as suggested by a novel methodology based on hierarchical clustering (Espinoza et al., 2012 ) that quantifies the numbers and sizes of clusters by computing an intrinsic distance. Two points belong to the same cluster if they are closer than this distance. This method was used in Figure 6C , where the clusters are enclosed by their convex hulls. Clustering analysis indicates that 456 out of the total 685 data points are in clusters, at the intrinsic distance d \n \n I \n = 12.8218, and the three largest cluster sizes are 13, 11 and 10. About 67.5% of the 456 extracted data points were in clusters of sizes 2–4, and there were 21 clusters of sizes at least 5. This is in line with observations that mono‐species biofilms of Salmonella tend to form scattered single‐cell or small clusters (Gonzalez‐Machado et al., 2018 ; Pang et al., 2017 ). FIGURE 6 (A) Confocal image (maximum projection Z‐stack) of replicate (3) from the most clusterable condition 1344 40 min NaClO 48 h. (B) 3D histogram representing the number of green channel particles extracted from z‐slices from the same condition with a maximum of 13 particles in a bin. (C) Plots of the clusters enclosed by their convex hulls, at the intrinsic distance dI = 12.8218, following the methodology developed in Unis ( 2010 ). (D) Ripley's K ‐function Kpois compared with estimates of the K ‐function based on different edge correction methods: Translation correction (Ktrans), Ripley's isotropic correction (Kiso), border correction (Kbord), using the Kest function from the spatstat R package. Deviations between the empirical K curves and the true value of K for a completely random (Poisson) point process, Kpois = πr \n 2 , may suggest spatial clustering Spatial point pattern analysis allows us to classify spatial distributions of the green channel particles in Figure 6A . A powerful tool for examining spatial independence across scales is Ripley's K ‐function which has been applied to quantify the spatial distribution of bacteria within the biofilm in several studies (Hart et al., 2019 ; Ishkov et al., 2021 ; Marchal et al., 2017 ). The univariate form of Ripley's K‐function (where only one type of point is considered) is. \n K ¯ r = A n 2 ∑ i ≠ j n w ij r I ij r , \n where n is the number of points inside a region of area A , w ij is the edge effect correction factor. The indicator function I ij defines whether a point p j is inside a neighbourhood r of point p i or not based on the Euclidean distance between the points p i and p j . Appropriate edge correction can improve the power of the statistical tests (Yamada & Rogerson, 2003 ). We used the spatstat R package ( https://cran.r-project.org/web/packages/spatstat/index.html ) which has different estimates of Ripley's K ‐function built in. Since these curves lie above the theoretical K ‐function K r = π r 2 , the point pattern in Figure 6D is clustered. Live and dead cells are not colocalized We used Imaris 14.0.0 (Bitplane, South Windsor, CT, USA) to quantify colocalization between live and dead cells on a given Z‐stackimage in order to understand whether distinct clusters differed in their chances of survival. The colocalization uses a statistical approach developed by Costes et al. ( 2004 ), which is done by estimating simultaneously the maximum threshold of intensity for the green and red channels below which particles exhibit no correlation. The main advantage of Costes method is that it automatically quantifies colocalization in any region of the image without user intervention. Imaris has been widely used to analyse image stacks for Salmonella in a variety of biofilm and immune host (Burton et al., 2014 ) settings. We used two main methods of colocalization analysis for the significant conditions, co‐occurrence and correlation. Co‐occurrence‐based colocalization analyses such as Mander's coefficient, determine the extent of spatial overlap between green and red fluorescent channels. Correlation‐based colocalization analyses such as Pearson's coefficient, describe the degree to which the abundance of the spatially overlapping channels are related to each other. Both approaches have different strengths and weaknesses (Aaron et al., 2018 ). Table S2 summarizes the Imaris output features for the clusterable confocal images such as quantification of colocalization between the two channels and for the individual channels green and red channel thresholds. There was no evidence of colocalization between the two channels, suggesting that live and dead bacteria establish spatially distinct regions within a Salmonella biofilm. This shows that clustering of survivors is not simply an artefact of where cells have produced most biomass. The colocalization of green channel particles alone was above 46% for all clusterable confocal images, except SL1344 20 min NaClO 24 h (2–3) and 3750 20 min PAA 24 h (1 and 3). The colocalization of the corresponding red channel particles was under 35%, except for SL1344 20 min NaClO 48 h (1) and SL1344 40 min PAA 24 h (2). The Pearson correlation coefficient (PCC) expresses to what degree signal intensity variation in green channel can be explained by the related variation in the red channel, assuming a linear relationship. Consistently medium‐to‐high positive PCC values were measured for SL1344 20 min H 2 O 2 48 h (1–3), SL1344 20 min NaClO 48 h (1–2), SL1344 40 min NaClO 48 h (1–3), SL1344 40 min H 2 O 2 48 h (1–3) cases (PCC >0.4). Interestingly, all these cases occur in SL1344 biofilms treated at 48 h with biocides of medium to low potency for 20–40 min. The Mander's coefficient accounts for the signal intensity of particles in each of the channels. M g is the co‐occurrence fraction of green colour with red colour and vice versa for M r . Except for SL1344 20 min NaClO 24 h (3) and SL1344 40 min PAA 24 h (2), all other clusterable confocal images have M g <0.24 and M r <0.19. These results indicate a low extent of co‐occurrence between live cells (and similarly, for dead cells) showing initial seeding is probably not related to final viability."
} | 9,020 |
33785764 | PMC8009962 | pmc | 6,050 | {
"abstract": "Benthic cyanobacterial mats (BCMs) are becoming increasingly common on coral reefs. In Fiji, blooms generally occur in nearshore areas during warm months but some are starting to prevail through cold months. Many fundamental knowledge gaps about BCM proliferation remain, including their composition and how they influence reef processes. This study examined a seasonal BCM bloom occurring in a 17-year-old no-take inshore reef area in Fiji. Surveys quantified the coverage of various BCM-types and estimated the biomass of key herbivorous fish functional groups. Using remote video observations, we compared fish herbivory (bite rates) on substrate covered primarily by BCMs (> 50%) to substrate lacking BCMs (< 10%) and looked for indications of fish (opportunistically) consuming BCMs. Samples of different BCM-types were analysed by microscopy and next-generation amplicon sequencing (16S rRNA). In total, BCMs covered 51 ± 4% (mean ± s.e.m) of the benthos. Herbivorous fish biomass was relatively high (212 ± 36 kg/ha) with good representation across functional groups. Bite rates were significantly reduced on BCM-dominated substratum, and no fish were unambiguously observed consuming BCMs. Seven different BCM-types were identified, with most containing a complex consortium of cyanobacteria. These results provide insight into BCM composition and impacts on inshore Pacific reefs.",
"introduction": "Introduction Though scarcely mentioned in the literature a decade ago, benthic cyanobacterial mats (BCMs) are receiving increasing attention from researchers and managers as being a nuisance on tropical coral reefs worldwide 1 – 4 . Whereas BCMs were known to bloom seasonally at some locations, the prevalence and duration of them are increasing at an alarming rate (see review 1 ). Though in part this observation can be linked to observers being more aware of them, a unique 40-year dataset from the Caribbean showed unequivocally that they have become more dominant in recent years alongside declines in hard corals and other key benthic groups 5 . The factors directly responsible for these changes remain uncertain but decreasing water quality and increasing water temperatures are likely primarily responsible 2 , 6 – 8 . Indeed, cyanobacteria are projected to become more problematic in a variety of aquatic systems in the coming years with increasing climate change-related factors and deteriorating local conditions that favour their growth 9 – 11 .\n Similarly to their better-studied freshwater counterparts 12 , proliferation of BCMs in coral reef ecosystems are associated with a wide range of problems due to their fast-growing opportunistic nature and their intrinsic toxic properties 1 . BCMs are notoriously competitive in interactions with corals, and often result in damage or mortality of coral tissue (e.g. Brown et al. 13 ). They can also overgrow other benthic species due to their fast growth rates and have been reported to smother organisms or cause stress due to the rich variety of secondary metabolites they produce 14 – 16 . BCMs can inhibit coral larval settlement and survival 17 , 18 , though effects appear to be species-specific, with high variation among different taxa (e.g. of corals, cyanobacteria) 19 . They are often reported to bloom during warmer summer months which commonly correspond to spawning times, when larvae are seeking suitable substrate to settle on. Given that successful coral recruitment and survivorship is critical for reef recovery dynamics 20 , BCM proliferation can be expected to seriously compromise the resilience of coral reefs to acute and chronic disturbances. They also release high amounts of bioavailable nitrogen 21 , 22 as many taxa fix nitrogen, and are one of the biggest benthic contributors of dissolved organic carbon 23 (DOC; i.e. sugars), thus further stimulating fast-growing primary producers and/or microorganisms and fuelling negative feedback loops 24 . Despite the recent surge in reports of BCM proliferation, there are still many gaps in basic knowledge, including their composition, the specific factors that facilitate their growth, and their impact on ecological processes and functions. Furthermore, in spite of the vast literature on reef fish diets and grazing preferences, little is known about BCM consumption by reef fish. In contrast, macroalgae (which have more commonly been implicated as the ‘villain’ on degraded reefs) have been comprehensively studied in terms of their effect on coral recruitment and survivorship 25 , 26 , outcomes of their interactions with hard corals 27 , 28 , and their palatability to a broad diversity of reef fish species (e.g. Rasher et al. 29 ). Accordingly, it is widely accepted that management of specific functional groups or species of fish that consume macroalgae can assist in their reduction (i.e. top-down control). The limited research into BCM consumption tends to indicate that top-down control of mats by fish is rather minimal (e.g. Capper et al. 30 ), implying that species-specific management would be futile in reducing them. Nonetheless, two recent observational studies have yielded some interesting insights into mat consumption. One study on Australia’s Great Barrier Reef observed Bolbometapon muricatum feeding on mats 31 , and another on the Caribbean island of Bonaire observed some species of angelfish ( Holacanthus tricolor, Pomacanthus paru ), parrotfish ( Scarus coeruleus, Scarus iseri ), and surgeonfish ( Acanthurus bahianus, Acanthurus coeruleus ), appearing to take bites from mats 32 ; though notably, ascertaining whether fish consume the cyanobacteria themselves, or are rather targeting trapped detritus, sediment, or other associated fauna is impossible through observations alone. While identifying reef species that may (opportunistically) consume mats is clearly important for management planning, of equal importance is understanding how BCM proliferation impacts reef fish processes at the reef scale. Herbivory is known to be critical for ecological resilience but is impeded at most tropical coral reefs due to human-mediated impacts such as overfishing and sedimentation 33 – 35 . Reefs in the Pacific Island region have much higher fish and benthic species richness than other areas such as the Red Sea and the Caribbean, facilitating higher functional redundancy (i.e. many species perform the same function 36 ) and greater response diversity (i.e. a large diversity of responses to ecosystem changes among species within a functional group 37 ), making this region particularly interesting to measure ecological responses to ecosystem changes such as BCM proliferation. Here, we investigate a natural BCM bloom at an inshore no-take marine protected area on Fiji’s Coral Coast. First, we quantified the benthic and fish communities at the study site during the seasonal BCM bloom and classified the different BCM-types observed based on their morphology and colouration. Second, we investigated how BCM proliferation impacted herbivory functions using remote video observations over natural grazable substrates dominated versus devoid of BCMs. Third, we assessed any indications of fish species (opportunistically) consuming BCMs from videos overlooking BCM-dominated substrate. Based on prior observations, we hypothesised that the presence of BCMs would impede herbivory and that BCMs themselves would not be consumed at a level that would indicate top-down control. Finally, we collected samples of dominant BCM-types and identified their cyanobacterial composition using a combination of microscopy and amplicon sequencing. To the best of our knowledge, this is both the first study into BCMs in Fiji and the first to document how BCMs impact fish herbivory in a natural, diverse in situ environment.",
"discussion": "Discussion This study measured the abundance of BCMs during a natural seasonal bloom on a Fijian inshore reef system, quantified how the BCMs influenced fish herbivory, and examined the cyanobacterial composition of the different BCMs present on the reef using a combination of microscopic and genomic tools. We found BCMs were the most abundant group, covering over half of the benthos during the sampled bloom, though throughout most of the year their cover is negligible (V. Bonito, personal observation). Seven different BCM-types were observed and described, each of which was found to have a unique and rich diversity of taxa within them. Video observations indicated that herbivorous fish grazing and scraping functions are significantly reduced where BCMs have overgrown grazable substrate and yielded no evidence of fish feeding directly on BCMs despite the diverse assemblage of fish present on the study reef. We hereby discuss the findings of this novel study of BCMs on Fijian reefs and in situ quantification of BCM effects on herbivory with a diverse, healthy fish community. Benthic surveys revealed that despite moderate coral cover and no macroalgae, BCMs were the dominant group within the benthic community at the time of the surveys, growing primarily over turf-dominated substrate. Importantly, over three-quarters of the grazable substrate (i.e. turf-covered rubble and pavement) of the reef was overgrown by BCMs. Aside from the BCMs, benthic composition at Namada was similar to that found at other nearby inshore reefs that have been protected from fishing pressure—well-grazed with minimal macroalgal cover, moderate hard coral cover, and turf algae as the dominant substrate cover 39 , 40 . Overall herbivorous fish biomass was ten-fold higher than a threshold identified below which shifts to algal-dominated reefs are more likely (i.e. 20 kg ha −1 ) 41 . With the exception of excavators, there was good representation of species across herbivorous functional groups. Video observations revealed that BCM-dominance impedes fish herbivory on the substrate. To our knowledge this is the first study that documents the impact of BCMs on herbivory functions in situ on natural substrates. Previous studies have assessed the palatability of BCMs in laboratories and/or have focused on multi-choice feeding assays or examining the effect of their extracted secondary metabolites 30 , 42 , 43 . Remote video observations were incorporated into this study for two reasons; (i) to see what impact BCM dominance has on detritivore, grazing, and scraping fish functions (i.e. those that target detritus, turf algae, and microphytobenthos) and (ii) to get an indication if any fish species were (opportunistically) consuming BCMs. Remote video observations offer a brilliant tool to assess the functioning of the fish community while avoiding the interference of any in-water divers, and recent studies have used them to quantify herbivory functions across different environments 44 , 45 . Importantly, our findings do not prove that no organisms feed on BCMs—this would need a different study design that also measures invertebrate grazers—but they do suggest that opportunistic consumption by herbivorous reef fishes is minimal. The impacts of BCMs on herbivory function will be dependent on the duration and extent of BCM blooms and the size of the reef. Our results do however suggest that as BCMs proliferate on reefs and expand their range, key ecological functions performed by herbivores could become impaired. Without a sufficient food source accessible in areas where BCM blooms persist, our results suggest that herbivorous fish would concentrate feeding on remaining grazable substrate, leading to higher competition for available resources, and potentially fish may ultimately be driven to leave and search for reef areas with higher food availability. Fish communities could also suffer directly from cyanobacterial blooms—a major die-off of juvenile rabbitfish Siganus argenteus and Siganus spinus attributed to starvation occurred as BCMs became dominant on coral reefs around Guam 46 . Reductions in diversity and abundance of fishes are known to be linked to coral mortality as a result of losses of topographic complexity and food availability among other factors, though herbivores have often been the most resilient to this due to constant or increasing availability of algae associated with coral decline 47 – 49 . If BCMs and not algae proliferate alongside coral loss, then impacts on herbivore communities may well be more conspicuous. Reductions in herbivory functions, even for relatively short periods of time, could result in turf algal communities developing into longer uncropped filamentous algae, similar to those observed in territorial damselfish territories 50 . While short well-cropped turfs can facilitate coral settlement and can thus be considered conducive to coral recovery 51 , long turfs significantly impede coral settlement 52 , and are more competitive in interactions with hard corals 40 . Furthermore, long turf assemblages are more likely to trap sediments and create negative feedback loops in herbivory 34 . Not only are herbivory functions critical on reef ecosystems, but herbivores also constitute an important part of human diets in regions such as that of the Pacific islands, and thus factors that affect herbivore populations are important to consider for the social consequences. Interestingly, when investigating the impact of bleaching-induced cyanobacteria proliferation on small coral-dwelling fish, researchers in Australia recently concluded there was no relationship between cyanobacteria and fish abundance at a local (1 m 2 ) scale 53 . Accordingly, we found no difference in the presence and activity of damselfish between BCM-dominated substrates and controls, indicating that at least short-term dominance of mats may not negatively impact small site-attached reef fishes. Some of these damselfish may even have been taking bites from mats, though gut analyses 54 would be required to determine whether they were consuming the cyanobacteria themselves rather than cleaning/removing detritus and/or sediment from their territory which we anticipate is more likely. Otherwise, no fish were observed to feed on BCMs during the video footage, suggesting that top-down control was limited at the study site. This is somewhat surprising given the high diversity of species detected in the study area, and the anticipated high response diversity to novel ecological states. As such we could have expected some species to opportunistically make use of the new dominant resource. These results also contrast to those of Cissell et al. 32 who identified several species to be consuming mats in the Caribbean. Reasons for these differences could include the presence of different herbivorous fish species, because of difficulties in visually determining what fish are feeding on, or differences in mat composition and chemistry. We identified seven distinct BCM-types at the study site and performed both microscopic and molecular analyses to gain first insights into the diversity they harbour. Both approaches have both their strengths and weaknesses, thus an integrated approach is recommended 55 – 57 . Traditional microscopic analyses have the advantage of requiring less sophisticated equipment but offer limited resolution because morphology can change with environment and distinction between species or genera is not always possible using morphological features. With molecular analyses, we are able to detect the presence of small cyanobacteria and distinguish between cryptotaxa, and thus generally identify more taxa than morphological analysis 58 , 59 . Examples of cryptotaxa include Lyngbya s.s . , Moorea, and Okeania— which are morphologically undistinguishable—as well as the genera that make up Leptolyngbya and Synechococcus 55 . On the other hand, morphotaxa have genetic uniformity and can only be distinguished by microscopy 57 . We used amplicon sequence variants (ASVs) rather than operational taxonomic units in this study as Knight et al. 60 recommend for microbiome analysis. Based on BLAST analysis (against the NCBI database) we could affiliate the ASVs to genera. The 16S rRNA dissimilarity-based identification of ASVs revealed that only four genera ( Moorea , Okeania , Lyngbya, and Oscillatoria ) met the criteria of a 95% cut-off of cyanobacterial genera delimitation that have previously been established (i.e. the best-blast hit with a similarity lower than 95% is probably not the genus that the ASV should be assigned to). Several ASVs annotated as Moorea producens , Oscillatoria nigro-viridis, and Okeania hirsuta met the 99% 16S similarity cut-off value to delimit species 38 , 61 . These findings are supported by the close proximity of these reference species to the associated ASVs in the phylogenetic tree (Fig. S2 ). However, we should be cautious with affiliating the ASVs to species level since the length of the marker gene that was sequenced was rather short (average 375 bp). All three are benthic marine species that have been found in tropical areas, e.g. O. nigro-viridis in Papua New Guinea 62 , M. producens also from Papua New Guinea but additionally found in the Caribbean 63 , and O. hirsuta in Okinawa 64 . As expected, our results highlight common discrepancies between microscopy and molecular analyses reported in many studies 55 , 56 , 58 , 65 – 67 . The microscopic analyses indicated that all samples except BCM-type E were dominated by Lyngbya s.l., and molecular analyses identified this further for three mats: the cryptotaxon Okeania dominated BCM-type F whilst the cryptotaxon Moorea dominated BCM-types C and G. Though the low number of taxa in BCM-types C and G observed in the sequence analysis was in agreement with the microscopy, the sequence analysis found the other BCMs to have a much higher diversity of cyanobacterial genera. Overall, ten genera were identified by microscopy compared to 18 by genomic analysis. Coral reef BCMs composed of diverse cyanobacteria taxa have also been reported in other recent studies that integrated molecular analyses based on 16S sequencing 22 , 68 . Furthermore, whilst the genera Oscillatoria , Spirulina , Anabaena cf., and Lyngbya s.l. (i.e. Moorea , Okeania, and Lyngbya s.s.) that were identified by microscopy were also detected by BLAST analysis, this was not the case for Calothrix , Phormidium , Tychonema , Leptolyngbya , and Heteroleibleinia , either because of the absence of these species in the reference databases or because of inaccuracies distinguishing them morphologically. Of the 18 ASVs detected in the sequence analysis, 14 of them showed a low similarity (< 95%) with affiliated genera indicating they could likely be new genera. For example, in BCM-types A, B, D, and E the contribution of a genus that was affiliated to Foliisarcina was quite high, but the affiliation to this genus had such a low similarity (~ 90%) that this ASV may be a new genus. The taxa identified as Anabaena cf. using both morphological and molecular approaches was previously unknown from marine ecosystems and could potentially be a new genus (Kaštovsky pers. comm. 2019). The clustering of the ASVs in the phylogenetic tree (Fig. S2 ) provides more information of other close lineages for the ASVs found in this study. Of course, the establishment of new genera requires further characterisation than minimal 16S dissimilarity criteria, but it suggests that tropical BCMs harbour a great deal of undiscovered diversity. As stated by Duperron et al. 38 , the 1700 described cyanobacterial species are only a subset of the actual diversity. Taxonomic studies of cyanobacteria have largely focused on freshwater pelagic cyanobacteria—which exhibit similar high diversity with many overlapping taxa to those identified here 12 —while tropical regions and marine benthic mats have been far less studied. New species and genera can be discovered in these habitats, which is of interest not only for the taxonomic but also chemical diversity 38 . Notably, it is also important to acknowledge that databases are incomplete and that low similarity may thus not necessarily reflect a novel genus and may be a result of the genus not being known to the reference database. Isolating and sequencing of cyanobacteria from mats would broaden our understanding of their composition, while the use of longer reads in sequencing would likely increase the resolution of the molecular methods 38 . It is very likely that the mats we sampled produce a high diversity of metabolites. Genera such as Lyngbya, Moorea, and Okeania are known for being chemically-rich in toxic compounds 63 , 69 . However, the chemical diversity that exists in other taxa that have not been taxonomically described yet remains largely unknown. Freshwater BCMs are better-studied thus provide some insights into the wide diversity and impacts of associated toxins 12 , and emphasise the need to expand research on marine BCMs. It’s likely that differences in the metabolites produced by different BCMs, along with variation in tolerance to the metabolites by different fish species, can in part explain discrepancies in observations of grazing on BCMs between this study and others (e.g. Cissell et al. 32 ). This study provided a preliminary description of the cyanobacteria composition of BCMs blooming at our study site in 2019; the first study of this nature on BCMs in Fiji. As the results reveal most BCMs consist of a diversity of taxa and likely identified several new genera of cyanobacteria, it would seem worthwhile to further extend this work with sampling at more sites, habitats, and depths to assess the taxonomic and chemical diversity of BCMs in relation to environmental drivers. More extensive morphological and molecular analyses would expand our knowledge of BCM composition. Furthermore, for proper characterisation of new genera and species, isolation of strains would be essential. A polyphasic approach 65 , incorporating morphological, molecular, and ecological data, is needed to describe cyanobacteria diversity and would facilitate more robust comparisons across studies. Despite the relative isolation of Pacific island reefs, they are by no means immune to the problems facing other tropical locations 70 . Coral cover is declining at many islands, with increasingly more reefs shifting into alternative regimes dominated by non-reef building organisms 71 , and BCMs are increasingly prevalent throughout the region as a result of iron input from shipwrecks, untreated sewage, and logging, among other factors 4 , 6 , 8 , 72 . For the last two years, mats were observed for the first time to endure throughout the colder months (May–August) at the study area. If BCMs become more of a permanent fixture at reefs, then our results suggest that herbivorous fish grazing will be concentrated in areas devoid of BCMs, potentially leading to increased competition for resources. We suggest that proliferation of BCMs on reefs may yield similar outcomes to increasing abundances of territorial damselfish due to reductions in herbivory functions and the creation of unfavourable biotic conditions for coral recruits. The potential for rollover effects on livelihoods and food security remains unknown. Finally, the results of this study further emphasise the need for reef surveys to differentiate between turf algae-covered substrate and BCM-covered substrate due to their vastly different ecological implications."
} | 5,859 |
29437248 | null | s2 | 6,051 | {
"abstract": "Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems."
} | 252 |
29437248 | null | s2 | 6,052 | {
"abstract": "Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems."
} | 252 |
26076480 | PMC4468158 | pmc | 6,055 | {
"abstract": "Rare earth elements (REEs) have become increasingly important metals used in modern technology. Processes including mining, oil refining, discarding of obsolete equipment containing REEs, and the use of REE-containing phosphate fertilizers may increase the likelihood of environmental contamination. However, there is a scarcity of information on the toxicity and accumulation of these metals to terrestrial primary producers in contaminated soils. The objective of this work was to assess the phytotoxicity and uptake from contaminated soil of six REEs (chloride forms of praseodymium, neodymium, samarium, terbium, dysprosium, and erbium) on three native plants ( Asclepias syriaca L., Desmodium canadense (L.) DC., Panicum virgatum L.) and two crop species ( Raphanus sativus L., Solanum lycopersicum L.) in separate dose-response experiments under growth chamber conditions. Limited effects of REEs were found on seed germination and speed of germination. Effects on aboveground and belowground biomass were more pronounced, especially for the three native species, which were always more sensitive than the crop species tested. Inhibition concentrations (IC25 and IC50) causing 25 or 50% reductions in plant biomass respectively, were measured. For the native species, the majority of aboveground biomass IC25s (11 out of 18) fell within 100 to 300 mg REE/kg dry soil. In comparison to the native species, IC25s for the crops were always greater than 400 mg REE/kg, with the majority of results (seven out of 12) falling above 700 mg REE/kg. IC50s were often not detected for the crops. Root biomass of native species was also affected at lower doses than in crops. REE uptake by plants was higher in the belowground parts than in the above-ground plant tissues. Results also revealed that chloride may have contributed to the sensitivity of the native species, Desmodium canadense , one of the most sensitive species studied. Nevertheless, these results demonstrated that phytotoxicity may be a concern in contaminated areas.",
"introduction": "Introduction Rare earth elements (hereafter referred to as REEs) are metals of the lanthanoid series in the periodic table. Though they are termed “rare”, REEs are in fact commonly found in soils worldwide [ 1 ]; the classification of “rare” solely refers to the lack of large deposits or ores that are characteristic of other elements such as silver and gold. Once widely mined, China’s low production cost for REEs in the 1990s significantly reduced the prices of REEs globally and consequently many other mines stopped extracting these valuable elements [ 2 ]. As a result, China currently mines and produces approximately 95% or more of the world’s supply of REEs [ 3 ], and thus has a monopoly on these critical resources. However, in recent years China has reduced its production and export of REEs to protect its industry and to decrease the environmental impacts that may result from REE mining [ 4 ]. To supplement their supplies, other countries including Canada and the USA (a former producer of REEs) have begun the process of researching, developing or reopening REE mining facilities. Of particular interest in North America are sites at Thor Lake, Northwest Territories [ 5 ] and Strange Lake, Quebec [ 6 ] in Canada, and the pre-existing REE mine at Mountain Pass in California, USA [ 7 ]. REEs are mined primarily for their usefulness in modern technologies, with many applications for green-technology developments [ 8 , 9 , 10 , 11 , 12 ]. Their most important usages are as components of high strength magnets in electronic equipment, in wind turbines and electric vehicles, for precision guided weapons, and in computers, audio equipment and automobiles, amongst others. They are also used in low quantities as fluid cracking catalysts during oil refining, in the production of optical glass and as components in phosphors for energy efficient lighting [ 11 ]. The risks of REE pollution due to mining and processing as well as from the improper disposal of materials containing these compounds could potentially lead to elevated levels within the environment. In addition, the processing of REE rich monazite rocks for the production of phosphate fertilizers and the subsequent applications of these fertilizers could further elevate REE soil concentrations, especially in agricultural areas [ 13 , 14 , 15 ]. In Canada, these fertilizers are commonly used within agriculture in the prairies, with application levels in the late 1990s reaching 775 000 tonnes per year [ 16 ]. Sneller et al. [ 17 ] reported that approximately 85 tonnes of neodymium (Nd) were released into the environment from phosphate fertilizer production in the Netherlands in 1994. Slooff et al. [ 18 ] reported that industrial emissions in air and water due to fertilizer production in the Netherlands can contain over 500 mg/kg of REEs. Petroleum refining processes can release similar amounts of REEs into the environment. In the USA, an estimated 60–80 tonnes of REEs per day are released into the atmosphere by oil refineries [ 18 ]. In a study conducted by Li et al. [ 19 ], it was found that REE soil pollution due to tailings from an REE processing plant in China can travel up to approximately seven kilometers before soil concentrations stabilize to natural levels. Specifically, Nd and praseodymium (Pr) levels in relation to the source were found to be, respectively, 5726 and 1614 mg/kg at 0.4 km, 2266 and 650 mg/kg at 0.8 km, 1279 and 373 mg/kg at 1.3 km, and 310 and 85 mg/kg at 2.1 km [ 19 ]. Slooff et al. [ 18 ] also report that soils in polluted sites near industrial locations in the Netherlands contain high levels of REEs [800–900 mg/kg cerium (Ce), 500–700 mg/kg lanthanum (La), 400 mg/kg Nd and 100 mg/kg Pr], which are at least 100 times higher than background levels. Concentrations in mining areas in China reached upwards of 200 times that of baseline earth crust levels for most REEs, including Pr, Nd and samarium (Sm) [ 20 ]. For these reasons, toxicity monitoring will become crucial as REE mining activities commence in Canada and other countries. Studies have indicated that REEs can be absorbed by plants due to the similar ionic radii that they share with calcium [ 21 , 22 ]. As a result, REEs may replace calcium molecules in a number of physiological processes involving proteins and enzymes, including root growth, photosynthesis, and flowering [ 15 , 21 , 23 , 24 , 25 ]. However, the mechanism of action of REEs in plants is still poorly understood [ 15 ]. Many studies have documented the presence of REEs in both the roots and shoots of a variety of different plant species; however, in these cases, the studies were conducted on plants growing in soils containing low, natural levels of the REEs [ 26 , 27 , 28 , 29 ]. Toxicological studies on the effects of REE soil contamination on plants are lacking, as the majority of research has been conducted under hydroponic growth conditions [ 21 , 30 , 31 ]. A wide range of reports from China, where REE fertilizers are regularly applied to crops, report stimulatory, positive effects of various REEs on different aspects of plant metabolism, growth and yield [ 22 , 32 , 33 , 34 ]. However, many of these positive effects are only observed at low doses of the REEs, with negative effects becoming apparent as dosages are increased [ 34 ]. Reported detrimental effects of elevated levels of REEs on plants include: decreased growth, root function and nutritional uptake [ 31 , 35 ]; reduced root elongation (erbium, Er [ 36 ]); decreased seed germination (La and mixed REE solution [ 37 ]); and chloroplast damage (terbium, Tb [ 38 ]). The importance of studying plants in the environmental assessment of contaminants can often be overlooked in favor of other organisms. For instance, Li et al. [ 19 ] observed that the soil macrofauna diversity near a REE processing plant in China was decreased at high REE concentrations, but unfortunately plant biodiversity and toxicity was not assessed. Plants, however, can serve as strong indicators of environmental health since they are literally grounded and cannot escape the presence of contaminants. In addition, since they are the primary producers of many ecosystems they serve as a major entryway for many contaminants into the food chain. For instance, Cowgill [ 39 ] observed uptake of several REEs by water-lilies ( Nymphaea odorata Aiton) and subsequently found these metals in aphids that fed on this species. In a previous paper, two light rare earth elements (LREEs) elements (La and Ce) as well as yttrium (Y) were examined for their toxicity to crops and native plants [ 35 ]. In this companion study we seek to assess the uptake and phytotoxicity of three light and three heavy rare earth elements (HREEs), on the germination and biomass of five plant species (three wild, native Canadian species and two crops) grown in soils contaminated with increasing concentrations of REEs.",
"discussion": "Discussion Due to the increasing worldwide demands for REEs in modern technology, global REE mining and extraction activities have been on a steady rise. Though they have become widely used, very little is currently known about the potential environmental impacts and toxicities of these elements to native plants growing in contaminated soils, thus warranting further environmental monitoring. Effects on germination Germination rates of all species were generally unaffected by REE soil concentrations. Some negative effects on speed of germination were identified for both R . sativus in Nd soils and S . lycopersicum in Er soils, but only at the highest dosage evaluated. Likewise, while effects were observed at intermediate doses for both R . sativus in Pr soils and D . canadense in Sm soils, the effect was not apparent at the highest dose. Due in part to these inconsistencies, it is not possible to rule out that these observed negative effects were due to variability in seed viability as opposed to REE toxicity. The results obtained in this experiment are similar to those found in a previous experiment with lanthanum (La), where no effects on germination were observed; however, the range of doses tested in the La study was significantly lower [ 35 ]. In contrast, two other REEs, yttrium and cerium, were observed to negatively affect the germination rates of the same species evaluated in the present study [ 35 ]. There is a paucity of studies on the effects of REEs on germination and early growth. Hu et al. [ 22 , 30 ] conducted studies in nutrient solutions rather than in soil and demonstrated both growth stimulation and inhibition. In contrast, D’Aquino et al. [ 37 ] observed reduced germination of Triticum durum Desf. seeds placed on germination paper after soaking in a La 3+ or a combined REE solution, with the observed negative effects varying by both seed soaking times (2–8 hours) and by concentration (0.01–10 mM). In the current experiment, when seeds were grown directly in REE contaminated soils, no significant stimulation in germination was observed for any of the species and REEs studied. Though the results indicate a potential slight toxic effect of some REEs on the seeds of certain species, the high soil dosage required to elicit these effects would be more representative of levels found at contaminated sites near mining or processing facilities [ 19 , 20 ], as opposed to sites with more subtle inputs (e.g. fertilized lands, landfill leaching). Effects on aboveground and belowground biomass High soil concentrations of the six REEs were needed to reduce the aboveground biomass of all plant species (as determined through IC25 and IC50 analyses). Native species were found to be significantly more sensitive to REEs (IC25s from 146.67 to 741.16 mg/kg dry soil) than crops (IC25s from 651.23 mg/kg dry soil to no effect). The same trend was observed for the IC50s. Likewise, effects on root biomass were more noticeable in native species than in the crops. Nevertheless, it is believed that significant quantities of REEs would have to be released into the environment to attain these potentially toxic levels. For instance, according to the information provided in Slooff et al. [ 18 ], the detected IC25 and IC50 values in this study are generally greater than the Nd and Pr soil concentrations reported at contaminated sites in the Netherlands (i.e. 400 mg Nd/kg soil and 100 mg Pr/kg soil), with the exceptions of the results for the three native species grown in Nd-spiked soils. In contrast to the HREEs (Tb, Dy and Er), levels of the LREEs (Pr, Nd and Sm) in contaminated sites in China exceeded both the EC25s and EC50s of most of the plant species investigated in this study [ 20 ]. Information on soil pollution levels of Sm, Tb, Dy and Er is scarce. Low to medium background concentrations of these REEs have been reported in natural soils, with high degrees of variability (0.51–20.93 mg Sm/kg soil, 0.13–2.3 mg Tb/kg soil, 0.51–12.1 mg Dy/kg soil and 0.16–6.2 mg Er/kg soil; see summaries in [ 54 , 55 ]). If a contamination factor of 100 times the background level is considered (as for Nd and Pr in the Netherlands [ 18 ]), it is likely that some plant species will be affected by these REEs; however, more site-specific baseline data from Canada and elsewhere around the world is necessary in order to form conclusions about their potential environmental impacts. In addition, further research on native species is required to properly measure hazards on other plant groups, such as bryophytes, pteridophytes, and woody plants. Chloride effect In most cases, D . canadense was the most sensitive species tested (four cases for both the IC25 and IC50). However, the lower values observed for this species may have partially been the result of the significant sensitivity of this species to the chloride molecule. Damage to one-year old avocado ( Persea americana Mill.) and citrus ( Citrus L. spp) plants in response to increased concentrations of chloride in irrigation water has been demonstrated in previous studies ([ 56 ] and references therein). In another experiment, it was shown that accumulation of chloride caused a reduction in gas exchange within leaves, as well as interacted with sodium to reduce other physiological processes in Citrus sinensis (L.) Osbeck cv. Hamlin seedlings [ 57 ]. The adverse effect of chloride associated with REEs had not been previously investigated. Our findings suggest that other experiments performed under hydroponic or soil conditions and which showed negative effects on plants [ 58 , 59 , 60 , 61 , 62 ] may be partly related to the chloride component of the tested compounds. Yet, undesirable effects of lanthanum have been observed with other REE forms [ 31 , 63 , 64 ], primarily with the nitrate form [ 30 ] that is more soluble than the oxide and the phosphate forms. Further research is thus needed to unravel the effect of REEs compared to their anions on plant phytotoxicity. Uptake and accumulation All species tested in this experiment were found to uptake and accumulate REEs from soils. Uptake and accumulation of REEs by the roots and shoots, respectively, was generally proportional to the doses tested. Accumulation into the shoots by the three native plant species (the most sensitive species) at the analyzed dose closest to the IC25 values also varied. The artificial soil used in this experiment was approximately pH = 6, representing a moderate value for Canadian soils that are more basic in the western Canada, but more acidic in the eastern Canada. In a previous experiment, it was found that at a lower soil pH (4.08), cerium (Ce) was more toxic to the majority of plant species than at a higher soil pH (6.74), and that Ce uptake by roots and accumulation in shoots was also generally much greater at lower soil pHs [ 35 ]. Tyler and Olsson [ 65 ] also studied the effects of soil pH on the uptake of metals by a grass, Agrostis capillaris L. For the lanthanoids, it was observed that root concentrations were inversely related to soil pH and positively correlated with soil concentration. Other research has indicated that soil pH plays a vital role in the bioavailability of various REEs and their release into soils [ 65 , 66 , 67 , 68 ]. It is probable that in a worse-case scenario, where soil has become acidic, REE contamination would become more toxic to plants growing at the site. As has been found in previous REE studies [ 27 , 35 , 42 , 65 , 69 , 70 , 71 ], accumulation of REEs was higher in the roots than in the shoots on a dry biomass basis. Specifically, Tyler and Olsson [ 28 ] found slightly lower concentrations of Pr, Nd, Sm, Tb, Dy and Er in the leaves of various species as compared to the roots. Fu et al. [ 27 ] observed concentrations ranging from 0.019 to 0.595, 0.001 to 0.097, and 0.0007 to 0.104 mg REE/kg dry weight in the roots, leaves, and stems, respectively, of ferns ( Matteuccia Todaro spp.) in soils containing approximately 0.191 to 1.697 mg/kg of the six REEs studied in this experiment. Those results, as well as those of Markert and Li [ 26 ], were in congruence with our detected plant concentrations at lower doses. Zhang et al. [ 71 ] found significantly higher concentrations of REEs in ferns than in other plant groups, as well as higher concentrations in leaves than roots, with root:soil and leaf:soil ratios much greater than those reported in this study (maximums of 7.26 (Sm) for root:soil and 16.07 (Nd) for leaf:soil). Wyttenbach et al. [ 72 ] observed Nd, Sm, and Tb leaf concentrations ranging from 0.033 to 0.544 mg/kg, 0.006 to 0.103 mg/kg and ~0.001 to 0.016 respectively in a variety of species grown in soils containing approximately 15.00 mg Nd/kg, 2.82 mg Sm/kg and 0.381 mg Tb/kg. Shoot to soil ratios varied between 0.002 ( Rubus fruticosus L. for Sm and Tb) to 0.044 ( Acer pseudoplantus L. for Tb) which is in concordance with the ratios observed for lower soil concentrations in our study (0.006 to 0.050). Another comprehensive experiment comparing 36 plant species grown in natural soils in Japan only detected REEs in 10 species, and only detected our six study REEs in up to three species: Dicranopteris dichotoma (Thunb.) Bernh., Athyrium yokoscense (Fr. & Sav.) C. Ch. and Phytolacca americana L.; however, detected soil concentrations were also low [ 44 ]. It was found that for Phytolacca americana , a species also native to Canada, leaf concentrations were sometimes higher than the corresponding soil concentrations [ 44 ]. On the high end, França et al. [ 29 ] measured Nd, Sm, and Tb leaf concentrations of tropical plants grown in Brazilian soils and detected REE concentrations ranging between 18 to 36 mg Nd/kg, 1.9 to 4.9 mg Sm/kg, and 0.24 to 0.47 mg Tb/kg to be < 1.5–28 mg Nd/kg, 0.019–4.2 mg Sm/kg, and < 0.009–0.24 mg Tb/kg respectively. As with several other studies, Pr, Dy and Er were not measured. Accumulation of REEs by plants at the lower doses in these experiments appears to correspond to rates reported for similar, natural REE soil concentrations. Unfortunately, information for higher soil concentrations is lacking. In all cases it can be seen that accumulation rates vary with plant group as well as with species; therefore, testing a variety of plants other than the commonly used crops is highly desirable. The REE plant:soil ratios, also referred to as transfer factors, in the present study were found to range from 0.001 to 0.278 for aboveground parts with only five of the 120 values above 0.100, with no immediate increase (hyper-accumulation) or decrease (plateauing) in absorption relative to the increasing soil concentrations. In contrast, the transfer factors of the aboveground system varied between 0.010 to 0.369 with 63 of the 120 values at or above 0.100. This is higher than values reported in other studies [ 26 , 41 , 73 ]; however, our control results were on par with both the leaf:soil ratios (average = 0.051 to 0.276 for Pr, Nd and Sm, respectively) and root:shoot ratios (average = 0.105 to 0.442) obtained where only background soil concentrations were considered [ 74 ]. There is no indication in the literature that REEs are essential to plants [ 20 ]. Some studies suggest that they may replace calcium and hence cause growth stimulation at low doses [ 66 ], although this has been disputed [ 30 , 31 ]. In China, Changle and Nongle, two commercial formulations comprising the nitrate form of REEs are used as seed treatment or sprayed at low doses on crops as fertilizers with demonstrated positive effects ([ 75 ] and references therein). Other studies conducted in hydroponic cultures have also demonstrated some growth stimulations. This was not found in the present study in plants grown in low dose contaminated soils. Conversely, higher soil levels of REEs that may arise in the vicinities of mining areas, landfills or where phosphate fertilizers are recurrently applied may be a concern for native terrestrial primary producers. The present study indicated that reductions in both aboveground and belowground biomass of wild native plant species did occur in the presence of elevated soil levels of REEs, therefore monitoring of sites near REE mines and processing facilities is of great importance."
} | 5,320 |
34063836 | PMC8224052 | pmc | 6,056 | {
"abstract": "Intensive exchange of nutrients is a crucial part of the complex interaction between a host plant and fungi within arbuscular mycorrhizal (AM) symbiosis. For the first time, the present study demonstrates how inoculation with AMF Rhizophagus irregularis affects the pea ( Pisum sativum L.) root metabolism at key stages of plant development. These correspond to days 21 (vegetation), 42 (flowering initiation), and 56 (fruiting-green pod). Metabolome profiling was carried out by means of a state-of-the-art GC-MS technique. The content shifts revealed include lipophilic compounds, sugars, carboxylates, and amino acids. The metabolic alterations were principally dependent on the stage of plant development but were also affected by the development of AM fungi, a fact which highlights interaction between symbiotic partners. The comparison of the present data with the results of leaf metabolome profiling earlier obtained did not reveal common signatures of metabolic response to mycorrhization in leaves and roots. We supposed that the feedback for the development and symbiotic interaction on the part of the supraorganismic system (root + AM fungi) was the cause of the difference between the metabolic profile shift in leaf and root cells that our examination revealed. New investigations are required to expand our knowledge of metabolome plasticity of the whole organism and/or system of organisms, and such results might be put to use for the intensification of sustainable agriculture.",
"conclusion": "5. Conclusions The data of our long-lasting investigation represent root metabolome alterations during host plant development. It has been clearly determined that transfer from vegetative growth, through flowering to fruiting, caused significant changes in the metabolic pathways that necessarily provided the required nutrients. The most intensive deviations occurred for sugars, lipophilic compounds, carboxylates, and amino acids. These data testified to the flexibility of metabolic networks specific to different stages of development. Besides that, rather intensive rearrangements were triggered by AM fungus interaction with pea roots. The appearance of different mycorrhizal structures enriches root metabolome profiles most probably due to the AM fungi metabolism and rearrangements of supply from pea leaves. Thus, root profile shifts result from a systemic metabolic response on the part of both host plant and symbiotic fungi. The most significant of these responses was related to lipophilic compounds, sugars, and amino acids. We suspect that the observed difference between the metabolic profile shift in leaf and root cells was caused by the feedback directed toward the development and symbiotic interaction of the supraorganismic system (root + AM fungi). Additional complicity of data analysis is also related to the well-known tissue specificity of metabolic pathways in different plant organs. The most important finding in our investigation is the estimation of the metabolic network adjustment for continuous developmental changes in pea plant roots hosting arbuscular mycorrhizal fungi. Both symbionts pass through physiological transformation and coordinate one another’s metabolism under unfavorable limitations in P supply. We have made only the first step in understanding the order of biochemical interactions and elucidating the signaling role of metabolites for mycorrhiza symbiosis. New investigations are required to expand our knowledge on metabolome plasticity of the whole organism and/or systems of organisms.",
"introduction": "1. Introduction Arbuscular mycorrhiza (AM) is a widespread symbiosis in nature formed by the vast majority of land plants with arbuscular-mycorrhizal fungi (AMF) of the Glomerymycota division [ 1 ]. AMF are obligate biotrophs: their life cycle is impossible without the supply of sugars and lipids from the host plant [ 2 , 3 , 4 ]. In plants, AM also performs several functions even though most plants can grow and develop without AM fungus in favorable conditions of mineral and water supply. AM thus improves plant water and mineral nutrition, including the uptake of inorganic phosphate (Pi), under natural conditions of strong competition for soil nutrients [ 1 , 5 , 6 ]. Likewise AM symbioses improve plant tolerance to both abiotic and biotic stresses [ 7 , 8 , 9 ]. Plant mycorrhizal dependency varies owing to the plant species/genotype, the type of fungus, the ecological adaptation of partners, and agroclimatic and soil conditions [ 1 , 6 , 10 , 11 , 12 ]. Therefore, in order to effectively apply plant symbiosis with AMF in sustainable agriculture, it is necessary to study in detail the ecological, genetic, physiological, and biochemical aspects of this interaction. Recently, a paradigm has been shifted from low-throughput, single end-point bioassays to systemic biology approaches. Thus, researchers have been employing so-called ‘omic’ tools, including transcriptomics, proteomics, and metabolomics [ 13 , 14 , 15 , 16 ]. Metabolomics is a particularly powerful tool for discovering how a plant’s physiological/biochemical status varies under different environmental conditions [ 13 , 14 ]. Numerous metabolomic studies revealed mycorrhiza-mediated changes in the biochemical composition of leaf tissues [ 11 , 17 ]. Some evidence for AM-induced root metabolome changes were shown for barrel medick ( Medicago truncatula ) [ 18 ], tomato [ 19 , 20 ], wheat [ 21 ], and soybean [ 22 , 23 ]. It is now known that the establishment of symbiosis with AMF affects the primary metabolism of the plant, thereby facilitating the exchange of photosynthates with AMF. The carbon supply leads to the transport of a significant amount of sugars to roots, as well as organic acids due to intensification of the Krebs cycle. Reprogramming secondary metabolite biosynthesis in mycorrhizal roots results in an increase in plant resistance to biotic and abiotic stresses. In addition, activation of the AMF phenylpropanoid pathway causes an increase in the diversity of secondary metabolites, a phenomenon which might serve to improve the quality of plant foods and pharmaceutical raw materials [ 24 , 25 ]. Especially noted was that the shifts in the plant metabolome triggered by symbiosis depend on both plant and AMF species. Therefore, an expansion of our knowledge of the metabolic changes within the interactions between different plants and AMF could allow for a more efficient use of this symbiotic association to increase plant productivity [ 25 ]. The pea ( Pisum sativum L.), an important legume crop, is able to form symbiosis with beneficial soil microorganisms, including AMF. Some pea genotypes exhibit relatively high mycorrhiza-dependence [ 26 ]. However, most of the studies conducted under model conditions [ 5 , 10 , 27 , 28 , 29 , 30 ], and in the field [ 31 ], indicate that the pea has a low responsiveness to mono-inoculation with AMF. Often, a noticeable positive effect of mycorrhization on the growth parameters of pea plants is observed only when they are grown under stressful conditions [ 27 , 29 ]. However, mycorrhization causes physiological and biochemical changes in the aboveground organs of pea plants [ 5 , 29 ]. In particular, Shtark et al. [ 5 ] showed that mycorrhization led to the retardation of plant development, which was also associated with an extended vegetation period and with an increase in the seed biomass of inoculated plants. Furthermore, during flowering and fruiting, the leaf metabolic profiles of inoculated pea plants shifted towards the profiles of the uninoculated plants at earlier developmental stages. A similar trend has been described for the seed proteomic profile in the pea line that had earlier been selected as highly sensitive to double inoculation with both AMF and nodule bacteria [ 26 , 32 ]. The line responded to inoculation by prolongation of seed maturation, manifested by up-regulation of the synthesis of proteins involved in cellular respiration, protein biosynthesis, and down-regulation of late-embryogenesis abundant protein synthesis. In contrast, the pea line with low responsivity to combined inoculation demonstrated lower levels of the proteins related to cell metabolism [ 32 ]. Thus, the analysis of the effect of inoculation with soil microorganisms on the physiology and biochemistry of pea plants, including the use of metabolomic profiling, can be useful for sustainable agriculture in the search for optimal technologies for the cultivation of different pea cultivars. At the same time, data about the effect of AM symbiosis on the pea metabolome are limited to a few studies focused only on changes in aboveground organs, such as leaves [ 5 , 29 , 33 ] and seeds [ 34 ]. There are no data on changes in the metabolome of pea roots under the influence of AMF. The aim of the present study was to analyze the effect of inoculation with AMF Rhizophagus irregularis on the root metabolome of pea plants at the key stages of plant development with application of gas chromatography-mass spectrometry (GC-MS). This paper presents the results of a comparative analysis of changes in the metabolite profiles of roots of non-mycorrhizal and mycorrhizal pea plants. Previously, AM-induced changes in the metabolite profiles of plant leaves from the same vegetation experiment were published in Shtark et al. [ 5 ]. Despite the fact that plants did not show a strong growth response to the inoculation, and their photosynthetic activity was not affected by mycorrhization, that study revealed significant metabolic alterations occurring in pea leaves during the AM development [ 5 ]. The present investigation showed that the pea root metabolome is very sensitive to AMF inoculation and depends on the stage of AM development. A comparison of AM-induced metabolic changes in the roots and leaves did not reveal common trends of metabolic response to mycorrhization.",
"discussion": "3. Discussion Plants have developed different adaptations to cope with unfavorable environmental conditions. Nutrient deficiency, particularly concerning soil-immobile elements such as P, usually trigger two events: changes in root architecture and establishment of arbuscular mycorrhizal (AM) symbiosis [ 35 , 36 ]. According to the literature, mycorrhization might result in the accumulation of proteins, carbohydrates, primary and secondary metabolites, probably owing to a better supply of phosphate and nitrogen [ 37 , 38 , 39 , 40 ]. Nonetheless, this accumulation does not always result in an increase of fresh weight. The low growth response of P. sativum to AMF mono-inoculation was shown in our previous research and some other publications [ 5 , 28 , 29 , 30 ]. Precise analysis of pea plant development during AM symbiosis revealed a very important event, namely that the pea plants inoculated with AMF prolonged the active phase of the vegetation period [ 5 ]. The effect was clearly distinguished with the application of GC-MS profiling of pea leaves. This investigation aimed to use the same experimental setup (host plant, AM inoculation, conditions of growing, etc.) to discover possible alterations in root metabolome under direct interaction with AMF. The pea root metabolomes obtained with GC-MS were less complex than those in leaves. A similar effect was earlier defined for other species grown under different environmental conditions [ 41 ]. Less diversity of carbohydrate metabolism in the roots is responsible for the lack of photosynthesis and associated carbohydrate metabolism pathways. Besides, limitation in monosaccharides in non-mycorrhizal pea plants might be linked to intensive exudation of primary metabolites into the rhizosphere. It involves different mechanisms, including passive losses and active exudation, but the exact way to regulate it is still poorly understood [ 42 ]. Plants are supposed to spend up to 20 to 40% of their photosynthetically fixed C in root exudates [ 43 ]. It is mainly suggested that exudates modify nutrient accessibility directly and through microbiota attraction. Some metabolites also might fulfill a regulatory role in plant development. For young plants, sugars efflux was shown to stimulate root elongation under P deficiency [ 44 ]. This might be the case for our data; under P-limitation at 21st DPI sugars were presumably transported out of the root system to facilitate root growth. At later stages of pea plant development, this effect disappeared. A large number of carboxylates (for example, malate, citrate, glycolate, succinate, malonate, etc.) were noted among the metabolites with a high content in young non-mycorrhizal plants ( Figure 2 ). These compounds are associated with various energy cycles that are in high demand at early stages of development. Carboxylates, especially citrate and malate, are also known to be secreted by the root system to facilitate nutrient absorption, including P, from poor soils [ 35 ]. Higher concentrations of different amino acids and amines most likely reflect intensive synthetic processes [ 23 ]. The latter are scarcely a required energy source and thus result in decrement to the levels of lipophilic compounds and sugars. Our previously obtained data highlighted a strong dependence on the part of leaf metabolite spectrum on plant development stages [ 5 ]. In this investigation, the same tendency was easily estimated with a simple PCA for root metabolomes ( Figure 2 ). The overwhelming majority of metabolites demonstrated non-monotonous alterations in pea root metabolic profiling. Sugars and lipophilic compounds were discovered to be the most variable. Most of the oligosaccharides, including sucrose, accumulated at 42 DPI, which was characterized with a transition to flowering ( Figure 3 ). Similar alterations were detected for lipophilic compounds like free fatty acids, sterols, etc. Preparation for flowering is a fundamental stage of plant development. The priority of different organs within the organism is supposed to be intensively revised and new cross-interactions and a sink/source relationship between plant organs have been established. The next stage tested, fruiting (green pod, 56 DPI), also caused alterations in root metabolome. Mycorrhization caused elevation of amino acid levels as well as the level of monounsaturated (18:1, 16:1) fatty acids. Along with metabolic rearrangement during pea plant development, a possible AM contribution was expected ( Figure 4 ). Pea roots were intensively sensitive to AMF inoculation. Mycorrhization at a vegetative stage resulted in higher accumulation of carboxylates and monosaccharides, while representation of lipophilic compounds (fatty acids, acylglycerols, sterols and other terpenes) were decreased. Multidirectional sets of changes were revealed for amino acids. Over transit to flowering, AM pea roots differed in the elevation of lipophilic compounds level and the expanding diversity of amino acids. At the beginning of fruiting, the inoculated pea roots still accumulated amino acids as well as monounsaturated fatty acid. Further enrichment analysis ( Figure 5 ) revealed successive changes of metabolic pathways from the turnover of carboxylates, pentoses, and intermediates of oxidative phosphorylation (21 DPI) to activation of fatty acid metabolism in mycorrhizal pea roots (42 DPI). At 56 DPI, enrichment analysis showed a decrease in the pathways associated with the exchange of fatty acids in the presence of the symbiont and intensification of amino acid and carbohydrates metabolism. The difficulty of evaluating the exact mycorrhization effect on the root metabolite profile is related to a complex interaction between plant and fungi. Actually, these biochemical alterations are the sum of such an interaction in the symbiotic pair. It has to be admitted that the intensive changes of AMF structures from penetrated hyphae to arbuscules, mycelium and vesicles are the result of different metabolic and physiological activities. Thus, the influence of AMF on metabolism in the examined profiles is quite possible. Furthermore, increased amounts of asparagine and aspartic acid in arbuscule-containing cells are associated with higher nitrogen availability. Arginine is shown to be desaminated in the intraradical mycelium and nitrogen is then transferred to the host plant in a form of ammonium [ 45 ]. The extent of the amino acid balance in inoculated roots is known to be very dependent on P supply [ 23 ]. We observed a weak elevation of the aromatic amino acid metabolism at early stages of vegetative growth. These are known to be precursors of phenolic compounds. AM induces stimulation of phenolic compound synthesis in roots that are known to positively regulate AMF spore germination, hyphal growth, and the colonization process [ 23 ]. AMF could be interfering with the sugar balance. The hexoses assimilated by AMF can further be transformed into trehalose and glycogen in the intraradical mycelium of AMF [ 46 ]. Recent investigations demonstrate that the AM fungus also obtains fatty acids from the host plant. They stimulated FA synthesis, transformed and deposited it as triacylglycerols, and transported it through mycelia to support AMF metabolism, growth, and sporulation [ 4 , 47 , 48 ]. It should be noted that metabolites establish clusters according to their chemical properties. The mapping of these metabolites was done according to strong correlations (|r| > 0.9, p < 0.01) with average levels of metabolites content in each experiment ( Figure 6 ). In the center of the obtained graph, the carboxylate single compact group can be distinguished with its attached amino acid and amine segment. Despite the importance of sugar levels and their alteration at different points of time, we did not find a single region for these metabolites. Weak connectivity of fatty acids with other metabolites could be explained on the basis of some metabolic apartness of lipid biosynthesis. This is clearly illustrated in Figure 5 , where pathways are mapped by the presence of common metabolites in metabolome. Weak connectivity between fatty acids and derivates is more intriguing, because they are closely metabolically linked. Just as for carbohydrates as for fatty acids, wide spreading on the graph may mirror the involvement of these metabolites in different biochemical processes with different functions. It is determined by more complicated quantitative regulation during processes of development and mycorrhization. The final step of our investigation involved comparing AM-induced metabolic changes in the roots (the model of this investigation) and leaves (the model of the previous one, [ 5 ]). For that we used the same time stages of pea plant development (21, 42, and 56 DPI). The PLS-DA models developed, and further comparison of differences between loadings of the predictive components, did not reveal a common schedule of metabolic response to mycorrhization in root and leaf ( Figure 7 ). To explain this fact we can only make the suggestion that the metabolic specificity between leaf and root is highly sensitive to the mycorrhizal symbiosis effect. Moreover, this phenomenon is a very complex, multicomponent reciprocal response which includes regulation of plant metabolism itself during development and demands of AMF. We know too little to assess the exact relationship between those processes yet. Differences in response to mycorrhization between roots and leaves are related to their metabolic distinctions. In particular, metabolites with a tendency to accumulate more in roots tend to be less stimulated by mycorrhiza ( Figure S5b ). From this we assumed that compounds supporting the growth of mycorrhizal fungus (especially amino acids) accumulate in the roots. At the same time, after the initiation of mycorrhiza growth, continuous consumption of these compounds is observed. This, in turn, strongly activates their synthesis in leaves, which is reflected in the rise of the level of such compounds."
} | 4,995 |
25653580 | PMC4299433 | pmc | 6,060 | {
"abstract": "Many of the precise biological mechanisms of synaptic plasticity remain elusive, but simulations of neural networks have greatly enhanced our understanding of how specific global functions arise from the massively parallel computation of neurons and local Hebbian or spike-timing dependent plasticity rules. For simulating large portions of neural tissue, this has created an increasingly strong need for large scale simulations of plastic neural networks on special purpose hardware platforms, because synaptic transmissions and updates are badly matched to computing style supported by current architectures. Because of the great diversity of biological plasticity phenomena and the corresponding diversity of models, there is a great need for testing various hypotheses about plasticity before committing to one hardware implementation. Here we present a novel framework for investigating different plasticity approaches on the SpiNNaker distributed digital neural simulation platform. The key innovation of the proposed architecture is to exploit the reconfigurability of the ARM processors inside SpiNNaker, dedicating a subset of them exclusively to process synaptic plasticity updates, while the rest perform the usual neural and synaptic simulations. We demonstrate the flexibility of the proposed approach by showing the implementation of a variety of spike- and rate-based learning rules, including standard Spike-Timing dependent plasticity (STDP), voltage-dependent STDP, and the rate-based BCM rule. We analyze their performance and validate them by running classical learning experiments in real time on a 4-chip SpiNNaker board. The result is an efficient, modular, flexible and scalable framework, which provides a valuable tool for the fast and easy exploration of learning models of very different kinds on the parallel and reconfigurable SpiNNaker system.",
"introduction": "1. Introduction Learning is crucial for the survival of biological organisms, because it allows the development of new skills, memories, and behaviors, in order to adapt to the information acquired from their local environment. Such high-level changes of behavior are the manifestation of an intricate interplay of synaptic plasticity processes, which lasts from early development throughout the adult life, and is taking place simultaneously and continuously in all parts of the nervous system. Although neuroscience has developed an increasingly better insight into the local plasticity mechanisms at specific types of synapses, we still have a poor understanding of the global effects of plasticity that lead to the emergence of our astonishing cognitive capabilities. Clearly, this is one of the great unsolved questions, not only for neuroscience, but with great implications for fields like philosophy, psychology, medicine, and also for engineering disciplines concerned with the development of artificial intelligent systems that can learn from their environment. Much of our understanding of the functional effects of local plasticity comes from theoretical and simulation studies of simplified learning rules in neural network models. Most influential is the hypothesis of Hebb ( 1949 ), which says that synaptic connections strengthen when two connected neurons have correlated firing activity. This has inspired many classical models for associative memory (Hopfield, 1982 ), feature extraction (Oja, 1982 ), or the development of receptive field properties (Bienenstock et al., 1982 ). Later, the discovery of Spike-timing Dependent Plasticity (STDP) (Markram et al., 1997 ; Bi and Poo, 1998 ) has led to a number of models that have exploited the precise timing properties of spiking neurons for receptive field development (Song and Abbott, 2001 ; Clopath et al., 2010 ), temporal coding (Gerstner et al., 1996 ; Guyonneau et al., 2005 ), rate normalization (Song et al., 2000 ; Kempter et al., 2001 ), or reward-modulated learning (Izhikevich, 2007 ; Legenstein et al., 2008 ; Friedrich et al., 2011 ; Potjans et al., 2011 ). It has also been realized that there is not one standard model for STDP, but that there is a huge diversity of learning rules in nature, depending on species, receptor, and neuron types (Abbott and Nelson, 2000 ; Kullmann et al., 2012 ), the presence or absence of neuromodulators (Pawlak et al., 2010 ; Cassenaer and Laurent, 2012 ), but also on other factors like post-synaptic membrane potential, position on the dendritic arbor, or synaptic weight (Sjöström et al., 2001 ). The discovery that basic effects can be achieved with local learning rules has had a big influence on the development of larger scale learning models that have mapped methods from machine intelligence onto spiking neural networks. Examples include supervised learning methods for classification of visual (e.g., Brader et al., 2007 ; Beyeler et al., 2013 ), or auditory stimuli (Sheik et al., 2012 ), and unsupervised learning methods like Expectation Maximization (Nessler et al., 2013 ; Kappel et al., 2014 ), Independent Component Analysis (Savin et al., 2010 ), or Contrastive Divergence (Neftci et al., 2014 ). This has opened up the possibility of using spiking neural networks efficiently for machine learning tasks, using learning algorithms that are more biologically plausible than backpropagation-type algorithms typically used for training artificial neural networks. The increased interest in spiking neural networks for basic research and engineering applications has created a strong interest for larger, yet computationally efficient simulation platforms for trying out new models and algorithms. Being able to easily and efficiently explore the behavior of different learning models is a very desirable characteristic of a such platform. The major problem for computation with spikes is that it is a resource-intensive task, due to the large number of neurons and synapses involved. Synaptic activity, and specifically synaptic plasticity, which might be triggered by every spike event, is dominating the computing costs in neural simulations (Morrison et al., 2005 ; Brette et al., 2007 ), partly because the communication and processing of large numbers of small messages (i.e., spikes), is a bad match for current von Neumann architectures. Different strategies to improve the scale and run-time efficiency of neural simulations either rely on supercomputer simulations (Plesser et al., 2007 ; Wong et al., 2013 ), parallel general-purpose devices such as GPUs (Fidjeland and Shanahan, 2010 ) and FPGAs (Neil and Liu, 2014 ), or special purpose neuromorphic hardware (Indiveri et al., 2011 ). Each solution involves a trade-off between efficiency, reconfigurability, scalability and power consumption. In this context we present a framework for studying arbitrary plasticity models on a parallel, configurable hardware architecture such as SpiNNaker. The SpiNNaker system (Furber et al., 2006 , 2014 ) has been designed as a massively parallel, highly reconfigurable digital platform consisting of multiple ARM cores, which optimally fits the communication requirements for exploring diverse synaptic plasticity models in large-scale neural simulations. Previous implementations of plasticity on SpiNNaker have been limited in their ability to model arbitrary spike- and rate-based learning rules. Here, we present a new approach for implementing arbitrary plasticity models on SpiNNaker, using a dedicated plasticity core that is separated from other cores that process other neural and synaptic events. Specifically we demonstrate the implementation of three synaptic plasticity rules with very different requirements on the trigger events, and on the need to store or access additional variables for computing the magnitude of updates. We show that the same architecture can implement the rate-based BCM rule (Bienenstock et al., 1982 ), an implementation of standard STDP based on a model by Morrison et al. ( 2008 ), and a voltage-dependent STDP rule suggested by Brader et al. ( 2007 ). We compare the efficiency and correctness of the STDP rule to previous implementations on SpiNNaker, and provide the first implementation of BCM and the learning rule of Brader et al. ( 2007 ) on this platform. All the experimental results presented in this paper come from implementations of learning rules on a 4-chip SpiNNaker board. The ability to implement different rules with very different requirements, that are either based purely on spike-timing, on the correlation of firing rates, or on additional voltage signals indicates that the framework can be used as a generic way of implementing plasticity in neural simulations. This new architecture therefore provides an efficient way for exploring new network models that make use of synaptic plasticity, including novel rules and combinations of different plasticity rules, and paves the way toward large-scale real-time learning systems. This article is organized as follows: the next Section introduces different approaches to model learning, from a theoretical and an implementation point of view. Section 3 describes the SpiNNaker system, the previous solutions for plasticity on SpiNNaker and our novel approach presented in this work. The flexibility of the framework introduced is demonstrated by the implementation of three different rules, presented in Section 4, 5, and 6: Spike-Timing Dependent Plasticity (Gerstner et al., 1996 ), the rate-based BCM rule (Bienenstock et al., 1982 ) and the voltage-dependent variation of the STDP rule (Brader et al., 2007 ). We validate the implementation by replicating classical plasticity experiments, and discuss the performances of each rule in Section 7. The paper is concluded in Section 8, which also provides an outlook toward future applications.",
"discussion": "7. Performance analysis and discussion In Diehl and Cook ( 2014 ), the authors describe an STDP variation of the DED which follows the strategy proposed in Morrison et al. ( 2008 ) by storing traces in SDRAM, rather than performing spike pairing as proposed in Jin et al. ( 2010b ). The authors evaluate the performance of their implementation as well as the one present in the stable release of the SpiNNaker software package 1 in terms of synaptic events processed per second, as done in Sharp and Furber ( 2013 ) and Stromatias et al. ( 2013 ). They do so by feeding a leaky integrate-and-fire population of 50 neurons with a neural population of variable size that produces spikes at ≈250 Hz, according to a Poisson process, with a 20% connection probability. They report that their implementation of plasticity is capable of handling around 500K synaptic events per second per core (using 150 input neurons), while the original SpiNNaker implementation is limited to 50K events. We adopt a similar strategy to evaluate our plasticity algorithms, but in more stringent conditions, and with a larger connectivity range. Rather than testing a single core we test a full chip (16 cores). In this way, we can also evaluate the effects of memory contention between different cores, as memory access can be a bottleneck for simulations on SpiNNaker. We model a population of 800 neurons in a single SpiNNaker chip (8 cores modeling neurons and 8 cores dedicated to plasticity) fed by an input Poisson neural population of 150 neurons with a variable rate, and measure the maximum firing rate at which the simulation can run in real time. We take as a starting point the connectivity levels reported by Diehl and Cook ( 2014 ) (20% interconnection probability, 150 × 50 × 0.2 synapses, for a total of 1,500 per core and 24,000 per chip if considering 16 cores) and increase the connectivity level up to 100% (7,500 synapses per core, 120,000 per chip). This results in synaptic rows which are 5 times longer, as every pre-synpatic neuron is connected to every post-synaptic neuron in each core, rather than only 20% as in the original experiment. We then scale the model further up by adding more pre-synaptic neurons so as to reach a total of 156,000 synapses. The performance analysis of the algorithms proposed in this work uses the same leaky integrate-and-fire current based neuron. To be able to scale the rate while maintaining the post-synaptic activity constant, we set all the weights and all the weight increments in the plasticity rules to 0, similarly to the approach in Stromatias et al. ( 2013 ). This means that plasticity is normally computed, but the weight is clipped to 0 and stored back in SDRAM. Such values are set at runtime and cannot therefore by optimized by the compiler; we have also ensured that setting these values to 0 would not bypass part of the code by removing some optimization tests (like not updating weights which do not change), thus ensuring that the code behaves in our test case as the worst possible real case. Post-synaptic activity is induced by feeding the leaky integrate-and-fire neurons with a current inducing an activity of ≈ 22 Hz. We check if at any moment any core is lagging behind real time as this would make the simulation incorrect and unrepeatable. We also check if a walk through of the weight matrix is completed before the end of the plasticity period or, in other words, if the plasticity process is finished before the next one starts, as overlapping in this sense is not possible when operating in real time. This allows us to measure the maximum number of synaptic events that can be handled in real time by a single SpiNNaker chip, using the three learning rules proposed in this paper (STDP, BCM and voltage-gated STDP), and to understand if the performance is limited by the neural or the plasticity core. Results are shown in Figure 11 ; for each given connectivity level (number of synapses) pre-synaptic firing rates are increased until the limit after which real-time simulation is no more possible. Each point of the plot hence represents the limits of the approach for a given connectivity, for each of the plasticity rules implemented. From the Figure it can be observed that the three learning rules implemented within this framework have similar performances untill the limit of 96,000 synapses (corresponding to scaling up to 80% connectivity the model by Diehl and Cook, 2014 ). This is due to the fact that, up to that point, all three learning rules are limited by the neural cores lagging behind real time, rather than by the plasticity process taking too long. Such limit peaks just below 1,5 million synaptic events per second per core for all three rules (23 million events for the full chip). In a non-plastic performance analysis, Stromatias et al. ( 2013 ) measured a maximum throughput of ≈ 2.38 million synaptic events per second per core. After this connectivity level the complexity of the two STDP models (standard and voltage-gated) becomes the limiting factor, and a complete walk of the synaptic matrix is not possible anymore within the 128 ms period used in this paper. The BCM algorithm is not affected by this, as the algorithm is computationally less intense, and keeps improving above 1,6 M synaptic events per second per core. The decay in performances reflects the complexity of the algorithm considered: standard STDP, being more complex than the voltage-gated version, has a sharper decrease in performances. Figure 11 Performance evaluation of the three learning rules in terms of synaptic event processed per core per second as a function of different number of synapses . When comparing these scenario results with the previous plasticity models based on the DED by Jin et al. ( 2010b ) and Diehl and Cook ( 2014 ) (around 50k and 500k synaptic events per second per core respectively in the 20% case - the leftmost part of Figure 11 ), it must be remembered that these algorithms work with a 1 ms spike-window resolution, while the experiments proposed in this paper have adopted a resolution of 2 ms. Also the former algorithms might lose spikes, while in the approach presented here the contributions from all the spikes are accumulated (or, in other words, no spike is lost). While our approach was designed for maximal flexibility, there might be tradeoffs in terms of efficiency for some scenarios, depending on connectivity and firing rates. One limitation of our approach is, for example, that every plasticity event triggers an update of the complete synaptic matrix. For the rules proposed in this paper is not possible to selectively update only some rows. For pre/post sensitive rules (such as STDP) destinations are encoded in the synaptic row, which is stored in SDRAM, so it is not possible to know if a pre neuron connects to a post neuron which has fired (thus inducing LTP) before retrieving the row itself. In rate based models such as BCM, where the firing rate is considered as a moving average, the absence of spikes is not sufficient to ensure there is no plasticity in act. Finally rules with relaxation toward one (BCM) or multiple (voltage-gated STDP) states require a weight update even in the absence of a spike. Since in our implementation plasticity updates occur every 128 ms, pre-synaptic firing rates should be at least on the order of ≈ 7 − 8 Hz to avoid having to update silent synapses regularly. In scenarios with lower firing rates, a purely event-driven update would be more efficient. However, a main motivation for our approach is to ensure real-time performance, even in situations with momentarily high load, e.g., if multiple neurons are firing in bursts. Such scenarios are common when using natural inputs with coincident input spikes or models with oscillatory background signals. In such cases the plasticity core approach offers greater flexibility to process plasticity in real time: instead of having to process neural and synaptic updates of all simultaneous spikes within the 1ms time step of the neural core, which might be challenging for complex plasticity rules or for complex neural models, our approach accumulates events over the longer time window of the plasticity core. This decoupling enables the neural cores to maintain the real-time constraints, and opens up new possibilities for trade-offs to reduce the load on the plasticity cores if necessary. The simplest possibility is, as in the DED model, to lower the number of neurons simulated by each neural core (and therefore also by its associated plasticity core). Other options, although not implemented in the first proof-of-concept presented in this paper, are possible. For the initial results presented in this work we maintain a 1:1 ratio between neural and plasticity cores, but this will likely not be optimal for all scenarios. When looking at Figure 11 it can be see that the two STDP models show a sweet spot for performance at around 80% connectivity. Before such maximum the performance is limited by the neural cores, while after that is the plasticity core which is not able to keep up with the real-time requirements. An interesting alternative would be to allocate more plasticity cores to a single neural core, and adapt the plasticity:neural core ratio according to the network characteristics and to the computational complexity of the neural and plasticity algorithms and the associated workload. A limiting hardware factor for any implementation of plasticity on SpiNNaker is the memory bandwidth, because rows of the synaptic matrix need to be written back to SDRAM. It was shown in Figure 9 of Painkras et al. ( 2013 ) that writing is the main bottleneck, since the read bandwidth is twice as high. Our approach reduces the write load, since rows are only written back to SDRAM at most once every plasticity interval, rather than once every pre-synaptic spike as in the DED model. This means that, for example, if pre-synaptic neurons are firing at 24 Hz each synaptic row would be transferred back to SDRAM 24 times per second using the DED model, but only 8 times with our approach. Finally another possibility is to increase the duration of plasticity intervals, which increases the time available for computing the updates, but comes at the cost of larger memory requirements for storing traces in the core-local DTCM. For long plasticity intervals this might grow beyond what can be stored in DTCM (64 Kbytes for each ARM core, of which some space needs to be reserved for other parameters and buffers). The capacity can be increased by lowering the precision for storing the traces, or using a coarser time resolution. All these possible trade-offs, although not fully explored in this initial work, show the versatility of the approach, which can be adapted to different situations and modeling needs, and constitutes one of its key features, as discussed in the last Section.\n\n8. Discussion Current research on understanding the relationship between the local electrochemical processes of synaptic plasticity and their manifestations as high-level behavioral learning and memory is increasingly relying on theoretical modeling and computer simulations (Gerstner et al., 2012 ). Because of the great diversity of plasticity phenomena observed in biology and the resulting diversity of proposed mathematical models, as well as the computational complexity of spiking neural network simulations dominated by the costs of synaptic processing, it is necessary to create simulations tools that provide both the flexibility to try out new models easily, and the speedup of specialized hardware. This meets the demand of increasingly large neural network simulations, both for studying brain function, and for applications in artificial intelligence (Le et al., 2012 ). SpiNNaker has proven to be a well-suited platform for massively parallel large-scale simulations of spiking neural networks, and is flexible enough to let researchers implement and test their own computational models in standard programming languages. The previous Deferred Event Driven Model of handling events in SpiNNaker has made it difficult to implement plasticity rules with arbitrary triggering events (pre-, or post-synaptic, or at regular time intervals), rules which depend on third factors available only at the post-synaptic neuron, or plasticity in networks with variable axonal delays. We have presented here a framework which uses the modular architecture of SpiNNaker and delegates weight updates to dedicated plasticity cores, while the network simulation operates on the remaining neural cores. We have shown that a variety of commonly used plasticity rules can be exactly replicated on this framework, with a greatly increased capacity of processing plasticity events in real-time, by running experiments on a 4-chip SpiNNaker board. The separation of neural from plastic concerns is the feature that enables the great flexibility of the architecture. The two cores work in parallel on different time scales and phases, and the plasticity core has all the information to compute plasticity for the recent past, can access the weight matrix shared with the neural core, and any other information that can be passed through means of shared memory, e.g., membrane potentials and spike timings of the pre- and post-synaptic neurons. All this information can be pre-processed before plasticity is computed, which allows e.g., the computation of rates in an otherwise spike-based simulation. The architecture can be configured easily, using PyNN scripts. This standard, high-level neural language makes it easy to integrate and explore new learning rules into the SpiNNaker architecture. The approach presented in this paper is tailored to SpiNNaker and to its specific architecture, design and constraints. Nonetheless the same principles could be applied to other digital-analog hybrid architectures, where efficient neural simulation could be realized on one neuromorphic chip, whereas complex plasticity rules could be realized off-chip on computers or FPGAs. Regarding GPUs it appears to be more favorable to let each kernel perform the same operation following the SIMD paradigm. Fidjeland et al. ( 2009 ) sequentially use two different kernels, one for neural updates and one for applying plasticity updates. Such kernels do not run in parallel on the same GPU, but serially. This does not constitute a problem when running accelerated simulations, which is the common case for GPUs, but can raise difficulties when running in closed-loop real-time scenarios, as in neurally inspired robotics (Galluppi et al., 2014 ). In fact concurrent kernel execution is a feature that has only recently been introduced in GPUs, with the NVIDIA Fermi architecture. Using such technique, a plasticity and a neural kernel could be instantiated concurrently on the same GPU, in a similar way to what is done in our approach. Memory access patterns, and the possibility of accessing contiguous portions of memory is a key factor when programming a GPU (Brette and Goodman, 2012 ). It could be speculated that applying an approach like the one proposed in this paper would have the benefit of guaranteeing memory coalescence, as the synaptic matrix is sequentially accessed when walking through it. Multi-core or cluster architectures could also in theory benefit of separating neural simulation and plasticity, running either on different threads or on different cores, and with different time scales. However, clusters are equipped with more powerful processing units than SpiNNaker, so computing neural and synaptic updates in different cores could introduce unnecessary overheads and synchronization difficulties, particularly regarding memory bandwidth and access patterns. In our experiments we have deliberately chosen to reproduce classical results, in order to compare the run-time performance of the novel framework to previous implementations of plasticity on SpiNNaker. The examples of BCM, STDP, and voltage-gated STDP learning provide templates for constructing further experiments with rate-based, spike-timing-based, and voltage-dependent learning rules. Our approach can be easily extended to include additional third factors to modulate plasticity, e.g., neuromodulators (Izhikevich, 2007 ; Potjans et al., 2011 ), or weight-dependency (Morrison et al., 2008 ; Nessler et al., 2013 ), can model homeostatic effects (Bartolozzi et al., 2008 ), or handle different synaptic delays (Tapson et al., 2013 ; Wang et al., 2013 ). It can also combine different models of plasticity in one simulation, a feature which is used in several recent models, where network function arises from the interaction of different synaptic plasticity rules that are specific to particular cell types (Lazar et al., 2009 ; Savin et al., 2010 ; Binas et al., 2014 ; Kleberg et al., 2014 ). In fact, we have provided a tool that should be general enough to model long-term potentiation rules, but is not restricted only to phenomenological ones. Other biological structures i.e., glial cells are considered to have a fundamental role in plasticity, and can enhance learning capabilities (Min et al., 2012 ). The plasticity core, by leveraging this functional segregation already present in biology, is a natural candidate to model such structures. The results presented in this work and the possibilities opened by this approach point to the efficiency and to the generality of the framework introduced: a modular, flexible and scalable tool for the fast and easy exploration of learning models of very different kinds on the parallel SpiNNaker system. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."
} | 6,926 |
35638897 | null | s2 | 6,063 | {
"abstract": "A novel composite hydrogel was developed that shows remarkable similarities to load bearing biological tissues. The composite gel consisting of a poly(vinyl alcohol (PVA) matrix filled with poly(acrylic acid) (PAA) microgel particles exhibits osmotic and mechanical properties that are qualitatively different from regular gels. In the PVA/PAA system the swollen PAA particles \"inflate\" the PVA network. The swelling of the PAA is limited by the tensile stress "
} | 115 |
31731432 | PMC6862615 | pmc | 6,064 | {
"abstract": "A superhydrophilic aluminum surface with fast water evaporation based on nanostructured aluminum oxide was fabricated via anodizing in pyrophosphoric acid. Anodizing aluminum in pyrophosphoric acid caused the successive formation of a barrier oxide film, a porous oxide film, pyramidal bundle structures with alumina nanofibers, and completely bent nanofibers. During the water contact angle measurements at 1 s after the water droplet was placed on the anodized surface, the contact angle rapidly decreased to less than 10°, and superhydrophilic behavior with the lowest contact angle measuring 2.0° was exhibited on the surface covered with the pyramidal bundle structures. As the measurement time of the contact angle decreased to 200–33 ms after the water placement, although the contact angle slightly increased in the initial stage due to the formation of porous alumina, at 33 ms after the water placement, the contact angle was 9.8°, indicating that superhydrophilicity with fast water evaporation was successfully obtained on the surface covered with the pyramidal bundle structures. We found that the shape of the pyramidal bundle structures was maintained in water without separation by in situ high-speed atomic force microscopy measurements.",
"conclusion": "4. Conclusions We investigated the superhydrophilicity of an aluminum surface anodized in pyrophosphoric acid. Pyrophosphoric acid anodizing leads to the fabrication of thin uniform oxide, porous oxide, pyramidal bundle structures consisting of many alumina nanofibers, and completely bent nanofibers. The change in the water contact angle with the anodizing time exhibits different behaviors depending on the measurement time after the water droplet is placed on the surface. During the measurement at 1 s after the water placement, the water contact angle greatly decreases to less than θ WCA = 10° by pyrophosphoric acid anodizing for up to 5 min, and a superhydrophilic aluminum surface is easily obtained. On the other hand, the contact angle slightly increases in the initial stage of anodizing due to the formation of porous alumina during the measurement at 200–33 ms after the water placement, and then, superhydrophilic behavior is exhibited on the nanofiber-covered surface. In particular, the value θ WCA = 9.8° at 33 ms after the water placement indicates that superhydrophilicity with fast water evaporation can be successfully achieved on the surface covered with the pyramidal alumina bundle structures.",
"introduction": "1. Introduction Aluminum possesses many attractive mechanical and physical properties, such as high strength-to-weight ratio, heat conductivity, electrical conductivity, and reflectivity; thus, aluminum alloys have been used for various industrial applications. Recently, wettability control on aluminum surfaces has been an important strategy for corrosion protection, heat exchange devices, and anti-icing surfaces [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. For example, a superhydrophilic aluminum surface with a water contact angle ( θ WCA ) measuring less than 10° exhibits rapid spreading of a water droplet, and such aluminum substrates have potential as high-efficiency plate-fin heat exchangers and antifouling materials [ 14 , 15 ]. Conversely, superhydrophobic aluminum surfaces measuring more than θ WCA = 150° with a low contact angle hysteresis exhibit excellent water-sliding behavior and have potential for self-cleaning materials and highly corrosion-resistant surfaces. Therefore, many surface finishing processes including painting [ 14 ], plasma treatment [ 16 , 17 ], colloidal coating [ 18 ], and carbon nanoparticle deposition [ 19 ] have been widely investigated for the fabrication of superhydrophilic and superhydrophobic aluminum surfaces. Electrochemical anodizing is one of the most important surface finishing processes for aluminum and its alloys and is widely employed for dielectric film coating [ 20 , 21 , 22 ], coloring [ 23 , 24 , 25 ], hardening [ 26 , 27 , 28 ], corrosion resistance improvements [ 29 , 30 , 31 ], and novel nanomaterial fabrication [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. So far, processes combining traditional anodizing in sulfuric, oxalic, and phosphoric acids with self-assembled monolayer (SAM) coatings have been reported by several research groups for superhydrophilic and superhydrophobic aluminum surfaces [ 44 , 45 ]. In this technique, anodic alumina nanofibers are formed on the surface via long-term anodizing and chemical dissolution of the upper part of porous alumina, and their nanostructure exhibits superhydrophilic behavior. In addition, subsequent hydrophobic SAM modification causes superhydrophobicity on the surface. However, it is difficult to accurately control the morphology of alumina nanofibers during anodizing due to the uneven chemical dissolution. Very recently, we have found the formation of alumina nanofibers formed by anodizing in a novel electrolyte, pyrophosphoric acid. Pyrophosphoric acid anodizing improves the controllability of the morphology of the alumina nanofibers due to the rapid dissolution of the anodic oxide and subsequent growth of the nanofibers during the initial stage of anodizing. Thus, highly ordered nanofiber arrays can be easily fabricated on the aluminum surface [ 46 , 47 , 48 ]. Superhydrophobic aluminum surfaces are successfully fabricated by anodizing in pyrophosphoric acid and with SAM modification [ 49 ]. Moreover, the fabrication of highly slippery and sticky superhydrophobic aluminum surfaces is easily achieved via the nanostructure control of alumina nanofibers [ 50 ]. On the other hand, the nanofiber-covered aluminum surface without SAM modification exhibits superhydrophilic behavior [ 51 , 52 , 53 ]. The contact angle measured on the superhydrophilic aluminum surface greatly changed with the anodizing time. In addition, it was observed that water evaporated quickly from the superhydrophilic surface. However, the effect of the morphology of the alumina nanofibers on the superhydrophilicity and corresponding fast water evaporation is still unclear. In the present investigation, we describe the superhydrophilicity on the aluminum surfaces covered with anodic alumina nanofibers and subsequent alumina bundle structures by anodizing in pyrophosphoric acid under the optimum operating condition. The superhydrophilic behavior was investigated by water contact angle investigation using a high-speed camera and water evaporation rate measurements. The morphology of the alumina nanofibers was examined by scanning electron microscopy and in situ high-speed atomic force microscopy, and a contact angle measuring less than 10° within 33 ms indicated that superhydrophilicity with fast water evaporation was successfully achieved by the formation of pyramidal alumina bundle structures on the surface.",
"discussion": "3. Results and Discussion The pretreatment specimens were anodized in pyrophosphoric acid at 80 V and 283 K for up to 180 min. Figure 1 a shows the change in the water contact angle measured on the aluminum specimen, θ WCA , with the anodizing time, t a . Here, the θ WCA values were measured at t d = 1 s after the 0.5 μL water droplet was placed on the surface. The electropolished aluminum surface exhibited hydrophilicity measuring θ WCA = 25.9° due to the formation of a hydrophilic, thin native oxide film after electropolishing [ 54 ]. As the electropolished specimen was anodized for 5 min, a superhydrophilic surface measuring θ WCA = 9.8° was successfully obtained due to the formation of anodic aluminum oxide. The θ WCA then gradually decreased with increasing anodizing time, and lower contact angles measuring 2°–4° were obtained after anodizing for 30–180 min. Notably, the minimum contact angle measuring 2.0° was obtained on the surface anodized for 60 min, and it appears that the contact angle very slightly increased with the anodizing time, although it is difficult to conclude a significant difference based on these small values. Therefore, the contact angles were also measured at the early stages after the droplet was placed on the surface using a high-speed CCD camera. Figure 1 b shows the θ WCA − t a curves obtained at t d = 200, 100, 66, and 33 ms after the 0.5 μL water droplet was placed on the surface. Although similar tendencies were obtained at t d = 200 ms through 33 ms, the contact angles obtained at each anodizing time decreased with increasing t d value. The minimum contact angle was measured at t a = 60 min in either case except for 66 ms, and superhydrophilicity, with a contact angle measuring θ WCA = 9.2°, was exhibited on the surface anodized for t a = 60 min within only 33 ms after the water placement. As the water droplet was placed on the superhydrophilic surface, the droplet spread isotropically and soon evaporated from the surface. Because the evaporation time of the water droplet strongly depends on the hydrophilicity of the surface, the evaporation time was investigated on the aluminum surface anodized for various operating times. Figure 2 shows the change in the evaporation time of a 0.5 μL water droplet on the specimen anodized under the same operating conditions shown in Figure 1 , t eva , with the anodizing time, t a . Here, the temperature of the aluminum substrate was maintained at 313 K using a thermoelectric Peltier module during the measurements. The time required for the complete evaporation of the water droplet from the electropolished surface was measured to be t eva = 88.5 s. The evaporation time decreased with the increased anodizing time, and the minimum rapid evaporation time measuring t eva = 26.3 s was obtained on the surface anodized for t a = 60 min. However, excess anodizing for more than 90 min caused the evaporation time increase to t eva = 29.2–41.6 s. Comparing Figure 2 with Figure 1 , the shape of this t eva − t a curve is in good agreement with the θ WCA − t a curves shown in Figure 1 . It is clear from Figure 1 and Figure 2 that the highest superhydrophilicity can be obtained on the aluminum surface anodized for 60 min. To investigate the effect of the morphology of the anodic oxide formed by pyrophosphoric acid anodizing on the superhydrophilic behavior, the surface of the anodized specimens was characterized by SEM. Figure 3 shows SEM images of the aluminum specimen anodized in pyrophosphoric acid at 80 V and 283 K for (a) 20 min, (b) 30 min, (c) 60 min, and (d) 180 min. As the electropolished aluminum specimen was anodized for 20 min ( Figure 3 a), a porous oxide film with numerous pores measuring 87 nm in average pore diameter was formed on the aluminum surface. Increasing the anodizing time to 30 min ( Figure 3 b) led to the growth of alumina nanofibers at the triple points of the honeycomb structure due to the chemical dissolution of anodic oxide during anodizing, and small pyramidal bundle structures consisting of several nanofibers were formed on the surface because of the bending and subsequent tangling of the alumina nanofibers. The number of alumina nanofibers contained in each bundle structure increased with the anodizing time due to the growth of the alumina nanofibers, and larger bundle structures were formed by anodizing for 60 min ( Figure 3 c). Further anodizing caused the formation of longer alumina nanofibers and subsequent complete bending due to their own weight, and the surface was covered with the bent alumina nanofibers ( Figure 3 d). Comparing the SEM images with the contact angle measurements and the evaporation behaviors, the following results are obtained: (a) Superhydrophilicity can be obtained on the porous alumina- and nanofiber-covered aluminum surface formed by anodizing in pyrophosphoric acid. (b) In particular, the highest superhydrophilicity is exhibited on the surface covered with the large pyramidal bundle structures after anodizing for 60 min. When the water droplet is placed on the aluminum surface covered with the pyramidal alumina bundle structures, one question is whether or not the shape of the bundles is maintained in water. To reveal the morphology of the alumina bundle structures in ultrapure water, the specimen anodized for 60 min was examined by in situ HS-AFM. Figure 4 a shows a HS-AFM image of a bundle structure formed by anodizing in pyrophosphoric acid at 293 K and 75 V for 15 min. It is clear that the bundle structures consisting of many alumina nanofibers maintained their shape in ultrapure water; the alumina nanofibers were not separated and fluttered in the underwater conditions. Figure 4 b,c show HS-AFM images of the bundle structure at 10 s and 20 s after starting the observation, respectively. Although several nanofibers were slightly moved by the scanning of the cantilever, the shape of the bundle structure and the alumina nanofibers was largely maintained during in situ HS-AFM observation for 20 s. Therefore, it is expected that the bundle structures maintained their shapes in water when the water droplet was placed on the anodized surface during the contact angle measurements. The reason that the bundle structures were unchanged in water may be due to the van der Waals forces between the alumina nanofibers [ 55 ]. Figure 5 a–c depict three-dimensional AFM images of a specimen anodized in pyrophosphoric acid at 283 K and 80 V for (a) t a = 20 min, (b) 60 min, and (c) 180 min, respectively. As the aluminum specimen was anodized for 20 min, a porous oxide film was formed on the surface ( Figure 3 a), and a relatively flat surface with numerous small nanopores was observed in the AFM image ( Figure 5 a). As the anodizing time increased to 60 min, many convex bundle structures of approximately 1 μm in maximum height were observed to be distributed on the surface ( Figure 3 c and Figure 5 b). However, further anodizing for 180 min caused the disappearance of the bundle structures due to the complete bending of longer alumina nanofibers, and the surface roughness decreased ( Figure 3 d and Figure 5 c). Figure 5 d summarizes the change in the arithmetic mean roughness measured from the AFM image, Ra, with the anodizing time. The roughness rapidly increased with the anodizing time during the initial stage due to the formation of alumina nanofibers and subsequent bundle structures, and a maximum roughness measuring Ra = 124 nm was obtained by anodizing for 60 min. With this anodizing time, the highest superhydrophilicity was exhibited on the aluminum surface ( Figure 1 and Figure 2 ). The roughness, then, gradually decreased to approximately 50 nm due to the bending of alumina nanofibers by excess anodizing. The water contact angle measured at this stage increased with the anodizing time. As compared the contact angle investigations with the surface roughness, the water contact angle and hydrophilic behavior clearly depend on the surface roughness. The contact angle of the droplet formed on the rough surface can be described by Wenzel’s equation as [ 56 ]\n \ncos θ W = R cos θ (1) \nwhere R corresponds to the specific surface area, and θ W and θ are the contact angles obtained on the rough surface and the flat surface, respectively. The anodic alumina nanofibers formed by pyrophosphoric acid anodizing consists of pure aluminum oxide without any electrolyte anion, and anodic aluminum oxide exhibits hydrophilicity due to the presence of the surface-bound hydroxyl groups [ 51 ]. Therefore, Wenzel’s equation indicates that the contact angle decreases with increasing specific surface area on such a hydrophilic surface. The aluminum surface covered with the pyramidal bundle structures consisting of numerous alumina nanofibers possesses a higher surface roughness ( Figure 5 d). In addition, SEM observation ( Figure 3 c) shows that many nanoscale spaces are formed under the pyramidal bundle structures. Moreover, strong capillary forces are induced by these alumina nanofibers. Therefore, the highest superhydrophilicity may be obtained on the surface covered with the pyramidal bundle structures by anodizing for 60 min ( Figure 1 and Figure 2 ). On the other hand, long-term anodizing leads to the disappearance of the bundle structures due to the complete bending of the long alumina nanofibers, and most of the aluminum surface is covered with the bent nanofibers ( Figure 3 d and Figure 5 c). Thus, the contact angle may slightly increase by excess anodizing for more than 60 min ( Figure 1 and Figure 2 ). Summarizing so far, the pyramidal alumina bundles are important nanostructures for the fabrication of superhydrophilic surfaces with fast water evaporation, but excess anodizing causes the hydrophilicity to decrease due to the disappearance of the bundle structures. On the other hand, short-term anodizing for 10 min caused a slight increase in the contact angle during the high-speed contact angle measurements for t d = 33–200 ms ( Figure 1 b), although the anodic oxide grew on the surface. Therefore, we demonstrate further contact angle investigations in the initial stage of pyrophosphoric acid anodizing in detail. Figure 6 shows the θ WCA − t a curves for the initial stage of anodizing on the aluminum surface anodized at 80 V and 283 K. At t d = 1 s after the droplet was placed on the surface, the contact angle rapidly decreased to θ WCA = 9.8° as the specimen was anodized for t a = 5 min and then slightly decreased with the anodizing time. However, very little change in the contact angle was measured during the range of 10 min < t a < 20 min. Based on the high-speed contact angle investigations at t d = 33–200 ms, it is noteworthy that the contact angle gradually increased with the anodizing time from 10 min < t a < 20 min, although hydrophilic aluminum oxide was formed on the surface via anodizing. There is a clear difference in the θ WCA − t a curves between t d = 1 s and 200–33 ms. Figure 7 shows SEM images of the anodized aluminum surface in the initial stage of (a) 1 min and (b) 10 min. A thin barrier oxide film with narrow stripes approximately 100–150 nm wide was observed on the surface anodized for 1 min ( Figure 7 a). This stripe pattern corresponds to the nanomorphology of the electropolished aluminum surface [ 57 ]. As the anodizing time increased to t a = 10 min, a porous alumina film with numerous nanopores measuring 52 nm in average diameter was formed ( Figure 7 b). Further anodizing for t a = 20 min caused the pore diameter increase by chemical dissolution of the anodic oxide (average pore diameter: 87 nm, Figure 3 a). Therefore, the period of the contact angle increase, t a = 10–20 min, corresponds to the expansion of the nanopores in the porous alumina matrix. To understand the effect of pore diameter in the porous alumina film on the contact angle, contact angle measurements were also performed using pore-widening porous alumina specimens. Here, the electropolished aluminum specimens were anodized in pyrophosphoric acid at 283 K and 80 V for 5 min to form a porous alumina film, and the anodized specimens were then immersed in a 0.52 M phosphoric acid solution at 293 K for up to t p = 15 min for pore-widening. Figure 8 a shows SEM images of the surface of the anodized specimen after pore-widening. Circular pores and linear trenches were formed on the surface by pore-widening for 2 min, and the porosity was calculated to be 22.8% using image analysis software. Although the porosity of the porous alumina was almost unchanged in a short immersion time for t p = 5 min, it gradually increased with immersion time by long-term pore-widening (25.2% for 10 min and 31.4% for 15 min). The porous alumina film was completely dissolved into the solution by immersion for 20 min. Figure 8 b shows the θ WCA − t p curves obtained on the anodized specimens after pore-widening. At t d = 1 s, the contact angle slightly decreased during the initial stage of pore-widening and then was almost unchanged during pore-widening. Conversely, it is clear that the contact angle obtained at t d = 33 ms gradually increased with the pore-widening time and corresponding the porosity. Based on Figure 6 and Figure 8 , the porosity—in other words, the diameter of the pores formed in the porous alumina film—strongly affects the contact angle obtained at the initial stage after the droplet is placed on the surface, and the contact angle increases with the pore diameter. When the water droplet is placed on the porous alumina film, water enters into the nanopores of the hydrophilic aluminum oxide. However, because a repulsive force is generated by the air in the pores, a composite surface consisting of aluminum oxide and air may be formed on the surface in the initial stage after the water placement ( Figure 9 a). The Cassie–Baxter model is widely employed to consider the contact angle formed on such a composite surface, and the contact angle obtained on the solid–air composite surface, θ c , can be described by the equation [ 56 ]\n \ncos θ c = f (1 + cos θ ) − 1\n (2) \nwhere f is the area fraction of the projected contact area and θ is the contact angle obtained on the flat surface. This equation indicates that a decrease in the solid area leads to an increase in the contact angle. The reason why the θ WCA obtained at t d = 33–200 ms increases with the anodizing time in the initial anodizing stage of t a = 10–20 min ( Figure 6 ) may be due to this relation. The contact angle increases with the anodizing time for up to 20 min because the diameter of the nanopores increases by pore-widening ( Figure 9 b). However, water enters into the nanopores of the hydrophilic aluminum oxide as the t d value increases, and the composite surface disappears from the surface. Thus, the contact angle slightly decreases with the anodizing time at t d = 1 s. On the other hand, the contact angles obtained at whole t d values decreased after anodizing for 30 min ( Figure 6 ). As described in Figure 3 , anodic alumina nanofibers and subsequent bundle structures were formed on the surface by anodizing for 30 min. The water droplet can quickly spread on the nanofiber-covered surface without closed nanopores due to the capillary effect of the nanofibers ( Figure 9 c); thus, superhydrophilicity with fast water evaporation appears on the surface after the formation of alumina nanofibers. By in situ HS-AFM, the shape of the pyramidal bundle structures was maintained in water without separation. Although several research groups have reported that superhydrophilic aluminum could be fabricated by anodizing, the time after the water droplet was placed on the surface during contact angle investigations was unknown in many cases. Ye et al. reported that ‘bird’s nest’ surface fabricated by typical anodizing in phosphoric acid exhibited superhydrophilicity measuring θ WCA = 3° when a water droplet was dropped on the surface after around 0.8 s [ 58 ]. In our investigation, the minimum contact angles measuring θ WCA = 2.0° at t d = 1 s and 3.0° at t d = 500 ms were obtained on the surface anodized in pyrophosphoric acid for 60 min, and this result is lower than that obtained by the previous investigation. Therefore, our anodizing technique using pyrophosphoric acid is useful for the fabrication of superhydrophilic aluminum surface in the various engineering fields."
} | 5,858 |
29404422 | PMC5781258 | pmc | 6,067 | {
"abstract": "The identification of a common “stress microbiome” indicates tightly controlled relationships between the plant host and bacterial associates and a conserved structure in bacterial communities associated with poplar trees under different growth conditions. The ability of the microbiome to buffer the plant from extreme environmental conditions coupled with the conserved stress microbiome observed in this study suggests an opportunity for future efforts aimed at predictably modulating the microbiome to optimize plant growth.",
"introduction": "INTRODUCTION The microbiome has the capacity to act as an extension of the host genotype that can respond to changes in environmental conditions and evolve rapidly ( 1 ). Changes in the host organism or environment have been shown to shift the composition of the associated microbiota in humans ( 2 , 3 ), mice ( 4 ), coral ( 5 ), and plants ( 6 ). The additional functions encoded by members of the plant microbiome can modify nutrient uptake ( 7 , 8 ), produce ( 9 ) or degrade ( 9 – 11 ) plant hormones, prime host defense pathways against pathogens ( 12 ) and pests ( 13 ), and ultimately affect both the above- and belowground growth of the host plant ( 14 – 16 ). In communities, effects of individual members can be additive ( 17 ), synergistic ( 18 – 20 ), or antagonistic ( 21 , 22 ). As a complex community, the plant belowground microbiome has been shown to adapt rapidly to water limitation conditions and alleviate host stress ( 23 , 24 ). This ability of individual microbes and microbial communities to modify plant growth characteristics and alleviate stress suggests an opportunity to optimize plant growth for biomass and yield, either by increasing plant growth and productivity or by modulating metabolite profiles for downstream biomass processing. Toward this goal, a thorough understanding of the plant-microbiome relationship along with the response of the microbiome to changes in the host and environment is required. Members of the Populus genus, fast-growing trees that are candidate second-generation biofuel feedstocks, are an attractive system for studying such microbiome intervention strategies. Recent 16S rRNA gene profiling studies of Populus growing in its natural habitat have contributed to our understanding of the Populus root microbiome community structure ( 25 – 28 ). These studies have demonstrated that Populus roots are host to a diverse bacterial community that differs on the basis of soil, geographic location, season, and host genotype but is most strongly influenced by the ecological niche (e.g., rhizosphere versus endosphere). Rhizosphere communities demonstrate an abundance of Acidobacteria that were greatly diminished in the endosphere (defined as within surface-sterilized root tissues), while Alphaproteobacteria and Gammaproteobacteria , along with Actinobacteria , were enriched in the root endosphere. Understanding the interplay between rhizosphere and root communities is critical for tailored microbiome intervention strategies to improve plant productivity. The production of biomass for food or energy crops will ultimately be affected by environmental conditions such as water and light availability and the presence of toxins. Climate change has led to changing precipitation regimes and more extreme weather events ( 29 ), and the ability of the microbiome to buffer against water limitation ( 23 ) provides an opportunity to mitigate the effect of these conditions in the field. Water limitation conditions have been shown to affect the microbiome by decreasing mycorrhizal colonization, ultimately decreasing nutrient acquisition by the host plant ( 30 ). Shading and cloud cover are natural limitations on light, decrease overall biomass production, and lead to structural changes in Populus species ( 31 ). These inhibitors have been shown to impact metabolite profiles in tomato ( 32 ) and tea ( 33 ) plants. Shading can significantly affect plant metabolite profiles and has been proposed as a method for optimizing secondary metabolite production ( 34 ). Shading, and the ultimate effect on plant photosynthesis and carbon allocation, also shifts the association of the plant with beneficial microbes in the environment ( 35 ). Finally, the presence of toxins and inorganic chemicals in the environment impacts the microbial community directly as antimicrobial compounds ( 36 ) or indirectly by either inhibiting the proliferation of other community members ( 37 ) or modifying the host exudate and chemical profile. Copper is an essential micronutrient for plants, but at high concentrations, it can inhibit plant growth in rice ( 38 ). In Populus , excess copper is accumulated in roots and decreases leaf chlorophyll content and photosynthesis ( 39 , 40 ). Responses to diverse stresses have been used to study the biology of bacteria ( 41 , 42 ), yeast ( 43 , 44 ), and plants ( 45 – 47 ). These studies identified core responses that were conserved across stress treatments which helped map the functions of genes and proteins in stress response. Gene expression studies show that while plants encode a wide range of mechanisms to deal with unique stresses, there is a subset of genes that are regulated in response to generic stress ( 45 – 51 ). For example, similar patterns of unique and core responses were observed in metabolite profiles in Zea mays (maize) responses to drought and heat stress ( 52 ). Similar to analysis of individual genes on host function, individual microbiome members have been shown to modulate gene expression on the basis of the presence of plant metabolites ( 53 , 54 ), suggesting a response to changes in the host metabolome, and indeed, the microbiome community has been linked to changes in the chemical environment of the host in engineered lignin mutants of Populus ( 55 ). In this work, we aimed to determine if a core response occurs in the microbiome of plants subjected to diverse stresses. Understanding how the plant and its associated microbiome respond to changes in the environment is critical for harnessing the protective and adaptive powers of the microbiome. We hypothesized that subsets of the plant belowground microbiome community (root and rhizosphere) would mirror the host response to stress, showing both a treatment-specific response and a core stress response showing microbial abundance changes that are shared between stress treatments. Using a microbiome inoculation strategy, we studied how Populus deltoides and its associated belowground microbiome respond to abiotic stresses of water limitation, shading, and copper toxicity.",
"discussion": "DISCUSSION In both natural and agronomic ecosystems, poor growth conditions can limit plant productivity and ultimately decrease the biomass yield. In this work, we present a systems level approach to the study of the phytobiome response to environmental treatments that induce plant stress. Using a belowground microbiome inoculation study and functional measures of plant growth, including gas exchange and fluorescence, plant transcriptional response, metabolite profile response, and microbiome community response, we show that the plant and associated belowground bacteria exhibit both stress-specific and core stress responses. This study suggests that the core microbiome members identified above appear to be tightly coupled to the physiology of the host plant and highlights the need for further testing to identify mechanisms of community change and consequences for phytobiome function and fitness. Plant responses, as measured by growth patterns, gas exchange and productivity, and leaf gene and metabolite expression profiles, indicated that plants were stressed in response to metal toxicity, water, and light limitation. However, the severity of the stress was likely different among copper, drought, and shade treatments. Consistent with previous studies, we observed decreases in plant productivity, shifts in gene expression toward the production of cell wall components, and decreases in photosynthetic processes ( 61 ) in water-limited plants. Previous work has shown that stress severity in drought treatments impacts the plant response at the physiological and gene expression levels ( 62 ), which presumably has subsequent downstream effects on the microbiome of the plant. The drought treatment in this study was cyclic, acute, and implemented in accordance with the individual plant response. With this approach, some plants experienced two drought cycles and others as many as five throughout the treatment period; however, a consistent response in terms of a microbiome effect was observed ( Fig. 4 and 5 ). There may be a differential response in plants maintained under long-term low-water conditions. Drought conditions affect both plant and soil environments, and these soil environmental effects likely contributed to the observed changes in the microbiome, especially in the rhizosphere ( 63 ). In contrast, the light limitation treatment is more specifically a host effect, limiting changes in the soil environment compared to water limitation. However, the reduced water requirements of shaded plants, as well as the decreased soil temperature owing to lack of direct sunlight may have affected environmental conditions and indirectly contributed to changes in the microbiome. In this work, copper stress was likely the least severe of the three treatments, as indicated by growth measurements and transcriptional responses. The lower stress severity may explain the observed weaker changes in the microbiome response. Conversely, the antimicrobial properties of copper may also have affected root-associated microbes directly or indirectly ( 37 ), which might contribute to the significant response in the gene expression results, which showed increases in the biotic stress pathway. Despite the diverse environmental changes imposed by these treatments, we did observe a common response in the microbiome community structure ( Fig. 4 and 5 ) that is best explained by the influence of the stressed host plant. The observation of a core stress microbiome was further supported by the results from a naive Bayes classifier in which stress samples that were incorrectly identified were primarily identified as one of the complementary stress treatments ( Data Set S1 ). The metabolic environment of the host contributes to the structure of the microbiome, either by modifying the metabolites available and the resulting competition or by direct inhibition of specific microbes. In water-limited plants, we observed consistent changes in metabolite profiles as consistent with other studies, which show increases in amino acids, phenolic compounds, and soluble sugars and sugar derivatives in leaves ( 64 – 67 ). Similarly, we observed changes in shaded plant metabolite profiles consistent with other studies of light limitation ( 32 , 33 , 68 ). In Scots pine ( Pinus sylvestris ) trees, shading leads to lipid-dominated respiration, as opposed to the carbohydrate-dominated respiration that is observed in water-limited trees ( 69 ). In Stellaria , shading changed the composition of gibberellins and auxin ( 70 ). Both shade and drought conditions have been shown to modulate carbohydrate, amino acid, and lipid contents in Pinus trees ( 69 ). However, it is not possible to conclude from this study or past studies whether changes in leaf metabolite profiles are a direct response to the environment and lead to changes in the microbiome or if the changes are a feedback result of changes in the microbial community. Plant metabolites have been shown to impact the microbial community ( 55 ), and inoculation by root microbes has been shown to impact leaf metabolite profiles ( 17 ), confounding the cause-effect relationship between the plant and its microbiome. Correlation analyses between OTUs and metabolites suggest that phenolic glycosides or other sugar conjugates may be driving plant-microbe interactions in this system, supporting the hypothesis that the plant host controls microbiome community members through differential feeding or inhibition of competitors. In addition, the identification of azelaic acid as highly correlated with gene expression data suggests some level of systemic resistance response to stresses ( 57 ), potentially contributing to the microbes associated with the plant. Anaerolineae , uncultured phyla, Rhizobiales , and Acidobacteriaceae OTUs were correlated with gene expression data, indicating a potential relationship between the microbiome response and the plant response ( Data Set S1 ). Further work is needed to elucidate the relationships among gene expression, metabolite production, and OTU abundances in order to understand and predict microbiome interactions with host plants. In this work, we studied the endpoint response of the belowground microbiome to plant stress. The analyses performed here and additional studies may enable strategies for controlling the microbiome to achieve reduced stress in plants. Of great future interest will be the dynamic response of the phytobiome to environmental stressors to determine both the time scale of functional responses and the implications for the microbial community associated with the plant. We did measure the photosynthesis kinetics of drought-treated plants and observed a functional response at the phytobiome level, but it is unclear how the microbiome responds during this dynamic time in the environment. Some microbes may be fast responders, on the order of hours, while others respond on the order of weeks. Additionally, the recovery of the plant microbiome as a community after host stress is unknown. Understanding how the microbiome rebounds after stress will also help us identity which microbes are important contributors to phytobiome function. Ultimately, it is the hope of the phytobiome community that we will be able to use this to harness the adaptive power of the microbiome and predictably modulate the system response. Our bacterial community results indicate that high-level taxonomy may be indicative of microbiome structure, with detailed functional changes attributed to specific OTUs. Despite changes in relative abundances and correlation structures, we did observe that high-level taxonomy (phylum and order) was similar between treatments and similar to other poplar ( 25 , 26 ) and other plant microbiome ( 71 ) studies. Uniqueness thus appears at lower taxonomic levels (family and below). This pattern may be associated with the broad phylogenetic relationship of complex phenotypes in the Bacteria kingdom. While some unique bacterial phenotypes are distributed within a phylum, complex phenotypes tend to be conserved at the family level or a higher level ( 72 ). Therefore, there is likely some commonality in the stressed environment or community that imposes the observed distribution of phyla in plant microbiomes. Further analyses identifying mechanisms leading to the observed stress response in the microbiome are required. In this work, we showed the response of the plant-microbiome system to diverse environmental conditions. Ideally, these results will inform future studies to generate and modulate communities with predictable and beneficial effects on the host plant."
} | 3,828 |
34123035 | PMC8145531 | pmc | 6,071 | {
"abstract": "As the smallest unit of life, cells attract interest due to their structural complexity and functional reliability. Protocells assembled by inanimate components are created as an artificial entity to mimic the structure and some essential properties of a natural cell, and artificial reaction networks are used to program the functions of protocells. Although the bottom-up construction of a protocell that can be considered truly ‘alive’ is still an ambitious goal, these man-made constructs with a certain degree of ‘liveness’ can offer effective tools to understand fundamental processes of cellular life, and have paved the new way for bionic applications. In this review, we highlight both the milestones and recent progress of protocells programmed by artificial reaction networks, including genetic circuits, enzyme-assisted non-genetic circuits, prebiotic mimicking reaction networks, and DNA dynamic circuits. Challenges and opportunities have also been discussed.",
"conclusion": "6. Conclusions and perspectives Protocells were initially created to study the origin and early evolution of life and to understand the mechanism of modern cells. Although both goals have not yet been achieved, numerous model protocells with cell-like structures and at least some of the essential properties of a natural cell have been developed and described in the present review. Rationally designed artificial reaction networks based on genetic systems were employed to reconstitute cellular functions in protocells. Complex behaviors, such as cell growth and division, metabolism, host immune response, and intercellular communication, were realized via enzyme-assisted or small molecule-based artificial reaction networks. DNA dynamic circuits were built on the surface or in the chamber of protocells as the computing core to construct cell-like automatons with a “logical mind”. As synthetic equivalents of natural cells, these properties or behaviors of protocells are modulated and controlled by equipping them with various artificial reaction networks so that future researchers can endow protocells with different functions in a customized way using programmed language and a bio-compiler. Although great achievements have been made, there are still many important challenges to be answered. One obvious limitation of the protocells reported is that almost all complex functions need external supplies of energy molecules or substrates and cannot sustain for a long time. Although the advancement of synthetic biology and technology allows the construction of more sophisticated bio-circuits, building more independent artificial reaction network propelled protocells is more profound and rewarding. Besides, it is the key to answer the question of the origin of life, since on primitive Earth no modern biomolecules existed. Although our attempts to generate an artificial entity able to evolve and be considered as a living cell have, thus far, failed, more rational hypotheses about the growth, division and replication of protocells on early Earth will be proposed. Another challenge is to segregate different artificial reaction networks effectively to afford protocells with organelles that operate spontaneously and even synergistically through the communication between these organelles. In addition, DNA reaction network-encapsulated artificial cells will attract increasing interest in the future because this strategy provides new principles of protocell construction and a biocompatible carrier or platform to build smart bionic automatons. A possible future direction is to integrate DNA circuits encapsulated inside a protocell with circuits built onto the protocell membrane so that DNA computation can be performed over the whole cell with enhanced programmability. Although this review has focused on the remarkable advances in the construction of protocells programmed from sketches of artificial reaction networks or a bottom-up approach to construct protocells, a top-down approach 100 that includes genetic manipulation or theoretical analysis of minimal genomes also shows potential in understanding the rules of cellular life. Perhaps developing a strategy that combines both top-down and bottom-up approaches will accelerate the studies of protocell construction and protocell-based biotics and bioengineering. Prospectively, prototissues 101 formed by interacting protocells that can sense and adapt to their surroundings will be the next stage of exploration and understanding of life.",
"introduction": "1. Introduction According to the Chemoton model propounded by Tibor Gánti, the primitive form of life should be as simple as possible, with only three fundamental features: metabolism, self-replication, and a bilipid membrane. 1 At the molecular level, metabolism, self-replication processes, and even membrane structures are organized and modulated by a series of spatiotemporally ordered chemical reactions termed chemical reaction networks. 2 The single cell is the basic structural and functional unit of living organisms, and its genetic and metabolic processes have been significantly studied since 1839. 3 One plausible way of understanding the mechanism of cellular life involves the assembly of inanimate components into artificial cells by creating artificial reaction networks. Since synthetic biology and chemistry are limited, these artificial reaction networks can program artificial cells 4,5 or protocells, 6,7 which mimic some natural cellular features. 8 However, if we consider protocells as cell-sized automatons with autonomous computing ability, then artificial reaction networks become the computational core or software of protocells, similarly to how natural reaction networks control the behavior, operation and communication of natural cells. Therefore, benefiting from the cell-like characteristics, protocell constructs can perhaps act as feasible candidates to work in biological circumstances with biologically and artificially combined algorithms and should find wide applications in some emerging fields such as biomedicine and bioengineering in the future. Some previous reviews have focused on the bionic features of protocells. 3,5 However, herein, we prefer to expand our review to include the various types of artificial reaction networks 9–11 used to build protocells. Accordingly, we will discuss four types of protocells in the following sections: protocells programmed by genetic circuitry, protocells programmed by enzyme-assisted non-genetic circuitry, protocells propelled by prebiotically mimicked reaction networks and protocells equipped with DNA dynamic circuitry ( Fig. 1 ). Specifically, protocells programmed by genetic circuitry and enzyme-assisted non-genetic circuitry are mainly constructed to study and understand the genetic and metabolic processes of modern cells through mimicry, while protocells propelled by prebiotically mimicked reaction networks are built to study the origin of the cellular system on primitive Earth. The purpose of constructing protocells equipped with DNA dynamic circuitry is different from the others because this field is focused on building cell-like automations. Because here we mainly focus on artificial reaction networks, top-down approaches 12 for manipulating genes in living cells to achieve different phenotypes are not discussed and some similar nomenclatures such as artificial cells, minimal cells, protocells and semi-synthetic cells are not differentiated in this review. For those who want a comprehensive understanding of these nomenclatures, the review by Caschera et al. 13 is recommended. Fig. 1 Schematic diagram of protocell programmed by various types of artificial reaction networks (ARN), including genetic reaction network, enzyme-assisted non-genetic circuit, prebiotically mimicked reaction network and DNA dynamic reaction network."
} | 1,964 |
30636412 | PMC6367683 | pmc | 6,072 | {
"abstract": "Stiffening due to internal stress\ngeneration is of paramount importance\nin living systems and is the foundation for many biomechanical processes.\nFor example, cells stiffen their surrounding matrix by pulling on\ncollagen and fibrin fibers. At the subcellular level, molecular motors\nprompt fluidization and actively stiffen the cytoskeleton by sliding\npolar actin filaments in opposite directions. Here, we demonstrate\nthat chemical cross-linking of a fibrous matrix of synthetic semiflexible\npolymers with thermoresponsive poly( N -isopropylacrylamide)\n(PNIPAM) produces internal stress by induction of a coil-to-globule\ntransition upon crossing the lower critical solution temperature of\nPNIPAM, resulting in a macroscopic stiffening response that spans\nmore than 3 orders of magnitude in modulus. The forces generated through\ncollapsing PNIPAM are sufficient to drive a fluid material into a\nstiff gel within a few seconds. Moreover, rigidified networks dramatically\nstiffen in response to applied shear stress featuring power law rheology\nwith exponents that match those of reconstituted collagen and actomyosin\nnetworks prestressed by molecular motors. This concept holds potential\nfor the rational design of synthetic materials that are fluid at room\ntemperature and rapidly rigidify at body temperature to form hydrogels\nmechanically and structurally akin to cells and tissues.",
"conclusion": "Conclusions We\nhave shown that chemical cross-linking of semiflexible PDA fibers\nwith a polymer exhibiting LSCT behavior endows the resultant hydrogels\nwith thermo- and stress-responsiveness. The coil-to-globule transition\nof PNIPAM-AC induces internal stress within the PDA semiflexible fibrous\nmatrix that drives the system into a stressed regime with an associated\nnetwork stiffening by up to 1000 times its room-temperature linear\nmodulus. The coil-to-globule transition of the linker rigidifies a\npreviously fluid PDA network to rapidly form elastic, strain-stiffening\nhydrogels. This holds promise in the biomedical field, where it opens\nthe door to their use as injectable materials that quickly form biomimetic\nscaffolds at body temperature provided that alternative strategies\nto cross-link PDA fibers avoiding the use of cytotoxic Cu(I) are explored,\nsuch as strain-promoted, cycloaddition reactions. 46 , 47 In addition, promising results have recently emerged making use\nof analogous bolaamphiphilic constructs as viable materials to support\nstem cell growth. 48 We have also\nshown that the so-formed hydrogels show quantitative\nresemblance to biological systems in the nonlinear stiffening regime\nfeaturing an exponential relationship of the differential modulus\nwith stress that directly mimics the stiffening of collagen gels and,\nmost notably, of reconstituted, active actin/myosin II networks and\nfibroblast subjected to mechanical prestress. Ultimately, we have\nillustrated the power and versatility of internally generated forces\nto enhance the mechanical response of soft materials constructed from\nentirely man-made building blocks, allowing us to emulate complex\nbiomechanical functions.",
"introduction": "Introduction Filamentous biomaterials,\nsuch as the actin cytoskeleton, collagen-based\nextracellular matrix, and fibrin blood clots, are three-dimensional,\ninterlinked meshworks of protein biopolymers. They are the scaffold\nof life, shaping and supporting our cells and tissues. In order to\ndo so in a robust and adaptive manner, their architecture (the spatial\narrangement of and connections between fibers) is highly dynamic,\nboth in terms of constituent polymers, which grow, shrink, and reorient, 1 − 3 and in terms of connections, which relocate, dissociate, and (re)bind. 4 − 6 Concomitantly, the mechanical response of a given architecture may\nbe actively amplified; previous work in cells, tissues, and reconstituted\nprotein meshworks has demonstrated the capacity of external and internal\nstresses and strains to change the stiffness of a material by orders\nof magnitude. 4 , 7 , 8 One\nsuch active control modality consists of the exertion of small and\nhighly localized forces on a polymer network. At subcellular scales,\nthese forces may be imparted by molecular motors; 9 − 13 in the extracellular matrix they arise from contractile\ncells (platelets and smooth muscle cells (SMCs)). 14 − 16 This microscopic\npinching is at the root of a number of highly functional biomechanical\nbehaviors: motors may prompt flow and fluidization of the cellular\ncytoskeleton to permit cell motility; 10 , 13 , 17 , 18 SMC-mediated forces\nexert significant prestress on the aortic wall, which strengthens\nit by prompting remodeling and deposition of additional collagen. 16 , 19 Platelet-mediated forces prompt the collapse and contractility of\nblood clots. Clearly, such responsive functionality allows biopolymeric\nmaterials to robustly perform and respond at different length scales\nand to a variety of external cues. Inspired by these biological\nregulatory mechanisms, recent work\nof Rowan and co-workers 20 successfully\nexploited the potential of lower critical solution temperature (LCST)\npolymers to augment the mechanical response of composite materials\nupon induction of coil–globule collapse. In their work, stiff\ncellulose nanocrystals (CNCs) were grafted with thermoresponsive poly(oligo(ethylene\nglycol)monomethyl ether (meth)acrylates) (POEG(M)A) and embedded within\na soft poly(vinyl acetate) (PVAc) rubbery matrix. Gels made from these\nmaterials reversibly change modulus with heating and cooling. Stiffening\narises from the formation of a percolated network of stiff CNC fibers\nbrought into physical contact via the collapse of the thermoresponsive\nelement, and softening to recover the original modulus is brought\nabout by rehydration of the collapsed globules below their LCST point. In this work, we combine the potential of polymers that exhibit\nLCST behavior to induce local contractile forces with the strain-stiffening\nresponse intrinsic to meshworks of semiflexible polymers. 21 We demonstrate that the induction of coil–globule\ncollapse of poly( N -isopropylacrylamide) (PNIPAM)\nchains that cross-link semiflexible fibers of poly(diacetylene) bis-urea\nbolaamphiphiles (PDA) ( Figure 1 ) dramatically changes the linear mechanical response, rigidifying\na previously fluid system to produce a robust and elastic material.\nMoreover, we show that in the nonlinear deformation regime universal\nstrain-stiffening occurs, with a power-law stiffening exponent that\nmatches that of collagen networks. This process happens at a constant\noverall volume. With this work, we engineer a strain-stiffening soft\nmaterial that shows a temperature-controlled rigidification induced\nby local strain. Figure 1 Molecules and methods used to construct biomimetic active\npolymer\nnetworks. (A) Molecular structure of the fiber-forming diacetylene\nbis-urea bolaamphiphile DA, its azide-functionalized analogue DA-N 3 , and the linear, thermoresponsive PNIPAM-AC linker. (B) Hierarchical\nself-assembly through intermolecular H-bonding and hydrophobic interactions\nof DA-N 3 and DA followed by topochemical polymerization\nof the assembled diacetylene groups into PDA fibers. Covalent fixation\nresults in strongly colored solutions due to the formation of a π-conjugated\nene-yne covalent framework. The fiber cross-section consists of 9\nor 10 ribbons of aggregated molecules. Chemical cross-linking with\nlinear PNIPAM-AC via CuAAC reaction into strain-stiffening networks\nwith triazole cross-links. (C) Internal stress generation within the\nfibrous PDA matrix mediated by PNIPAM-AC coil-to-globule transition\nabove its LCST. (D) Cryo-electron micrograph (cryo-EM) of PDA fibers\nin water (1 mM). Scale bar: 200 nm.",
"discussion": "Results and Discussion In previous work, we introduced diacetylene\nbisurea bolaamphiphiles\n(DA) ( Figure 1 A) as\na versatile motif to construct strain-stiffening hydrogels. 22 In water, DA molecules self-assemble through\nan interplay of intermolecular urea–urea hydrogen bonds (that\nguide the 1D assembly process) and hydrophobic interactions into semiflexible\nfibers ( Figure 1 D).\nThe so-formed fibers can be mechanically reinforced with covalent\nbonds via photopolymerization of the assembled diacetylene groups\ninto PDA fibers with an associated change in optical properties owing\nto the formation of a π-conjugated framework. 23 In aqueous media, PDA fibers have an average contour length\nof 157 nm, persistence length of 280 nm, and a cross-sectional diameter\nof 3.3 nm. 22 Further analysis revealed\nthat PDA fibers’ cross-section contains 9 or 10 ribbons of\naggregated molecules ( Figure 1 B), thereby imparting the bending stiffness needed to be applied\nas protein mimics. Gelation of PDA fibers was achieved by introducing\ncross-linkable analogues (DA-N 3 ) into the fiber-forming\nDA host before the covalent fixation step. Thus, clicking of azide-\nand acetylene-labeled PDA fibers by means of a ligand-accelerated\nCu-catalyzed cycloaddition reaction (CuAAC) 24 yielded strain-stiffening gels without an increase in fiber dimensions\n(i.e., no additional bundling) upon chemical cross-linking. 22 In the current work, PDA fibers functionalized\nby incorporation of 20 mol % DA-N 3 were chemically cross-linked\nwith a PNIPAM copolymer containing 5% propargyl acrylate residues\n(PNIPAM-AC). The reaction afforded fibrous gels covalently interlinked\nwith a thermoresponsive linear polymer as schematically depicted in Figure 1 B. Synthesis and Characterization\nof PNIPAM-AC PNIPAM-AC\nwas prepared via reversible addition–fragmentation chain-transfer\n(RAFT) copolymerization of NIPAM and trimethylsilyl (TMS)-protected\npropargyl acrylate. TMS protection was carried out in order to prevent\nunwanted branching and eventual cross-linking of the individual chains\nby polymerization of the somewhat polymerizable terminal alkyne moieties. 25 In the final step, the TMS groups were removed\nwith tetra- n -butylammonium fluoride (TBAF) to give\na linear polymer with an average molecular weight of M n = 6.96 kDa and a dispersity of Đ M = 1.08 (see Supporting Information ). Thus, each polymer chain consists of 62 repeat units on average,\nof which 3.1 are propargyl acrylate residues. The cloud point of PNIPAM-AC\nin water was studied by measuring the transmittance at 600 nm in a\nUV–vis spectrophotometer over a temperature range from 20 to\n35 °C. Solutions became turbid at ca. 27 °C at a concentration\nof 5 mg mL –1 , and the drop in transmittance shifted\ntoward lower temperatures at increased polymer concentration ( Figure 2 ). The cloud point\ntemperature was taken as the temperature at which transmission had\ndropped by 50% ( T CP(50%) ) and was lower\nthan the literature value of 32 °C, 26 likely due to the incorporation of a hydrophobic co-monomer, as\nhas been previously reported for other PNIPAM copolymers. 27 , 28 Increasing the amount of propargyl acrylate to 10 mol % in the monomer\nfeed rendered the polymers insoluble in water, which limited the degree\nof functionalization of PNIPAM-AC. Figure 2 Cloud point temperatures ( T CP(50%) )\nof PNIPAM-AC coils in water measured at different polymer concentrations. Gelation and Thermal Analysis\nof the Hydrogels Chemical\ncross-linking was initiated by adding the catalyst mixture to an aqueous\nsolution of polymerized PDA (containing 20 mol % DA-N 3 )\nfibers and PNIPAM-AC. Solutions were immediately transferred to the\nrheometer, where the gelation process was monitored by measuring the\nchange in moduli at a constant temperature of 20 °C with small-amplitude\noscillatory strain (1%) until a constant value of the elastic modulus G ′ was reached ( Figure S3 ). Concurrently, networks below the critical connectivity\nthreshold (no measurable storage modulus G ′ at 20 °C) were allowed to react for ca. 10\nh in the rheometer prior to analysis. Thermal analysis was performed\nby subjecting the hydrogels to a linear temperature ramp from 20 to\n55 °C while continuously recording the change in moduli with\nsmall-amplitude oscillatory strains. At room temperature, increasing\nthe concentrations at a fixed ratio of acetylene to azide groups,\n[PNINAM-AC]/[DA-N 3 ] = 0.78, produced progressively stiffer\nmaterials with moduli G ′ ranging from 2 to\n200 Pa in the concentration range between 13 and 23 mg mL –1 ( Figure 3 A). Below\n10 mg mL –1 PDA however, the storage modulus of the\nnetworks could not be probed at room temperature (yellow region of Figure 3 A). Within this concentration\nregime, all networks remained in the liquid state below the LCST of\nPNIPAM-AC and formed hydrogels able to support their own weight after\nplacing the sample tubes in a hot water bath at 55 °C. Rigidification\nof the gels took place within seconds without macroscopic shrinkage,\nnor was water expelled from the hydrogel ( Figure S4 ). Samples remained in the gel state even weeks after returning\nto room temperature. Rheology during the T -ramp showed\nan increase of G ′ by more than 2 orders of\nmagnitude with the stiffening setting in at temperatures slightly\nabove the measured cloud point of PNIPAM-AC in water ( Figure 3 A) and gradually shifting toward\nlower temperatures at higher polymer concentrations. In line with\nthis observation, although “free” PNIPAM-AC exhibits\na characteristic concentration-dependent shift in cloud point, this\neffect was enhanced in the presence of PDA fibers screening the globules.\nFor instance, while a 5 mg mL –1 PNIPAM-AC solution\nbecame turbid at 25 °C (in Figure 2 ), the transition was shifted to ca. 35 °C for\nthe same concentration of PNIPAM-AC when cross-linked to 10 mg mL –1 PDA fibers (blue up-triangles in Figure 3 A). Although kinetic effects\ncan be excluded as the main source of this delayed cloud point—given\nthe fast rigidification observed when samples were placed in a hot\nwater bath compared to the experimental time scale of the T -ramp, i.e., 28 min—similar shifts in LCST have\nbeen reported in thermoresponsive POEG(M)A polymers when grafted to\nCNCs and have been ascribed to the hydrophilic nature of the CNC. 20 Figure 3 Thermal stiffening of PDA/PNIPAM-AC hydrogels. (A) Linear\nstorage\nmodulus G ′ vs temperature\nrecorded by applying γ = 1% and ω = 6.28 rad s –1 at a linear heating rate of 1.25 °C min –1 for different PDA (20 mol % DA-N 3 ) concentrations cross-linked\nusing a fixed molar ratio of acetylene to azide groups, [PNIPAM-AC]/[DA-N 3 ] = 0.78. The yellow region represents the concentration threshold\nrequired for connectivity at 20 °C. (B) G ′ vs T for 15 mg mL –1 PDA (20 mol % DA-N 3 ) hydrogels cross-linked using different\n[PNINAM-AC]/[DA-N 3 ] molar ratios. The significant increase in G ′ prompted by PNIPAM-AC coil-to-globule transition seen in Figure 3 can be related to\nthe isotropic nature of the induced deformation, whereby PNIPAM-AC\ncollapse pulls on PDA fibers regardless of their initial orientation.\nBy contrast, stiffening due to anisotropic shear stress preferentially\nrecruits fibers aligned in the direction of the imposed strain. 21 , 29 , 30 The approximately 100-fold increase\nin modulus found for the PNIPAM-containing system is reminiscent of\nfilamin A (FLNa)-cross-linked F-actin networks isotropically stressed\nvia contractile forces imparted by embedded myosin II motor proteins\nor of fibrin in blood clots stiffened by contractile platelet-mediated\nforces. 12 , 13 , 31 − 33 To study the effect of the ratio of acetylene to azide groups\non\nthe macroscopic properties of the hydrogels, solutions containing\n15 mg mL –1 PDA fibers (containing 20 mol % DA-N 3 ) were cross-linked using varying concentrations of PNIPAM-AC\nranging from 1.5 to 15 mg mL –1 , resulting in acetylene\nto azide ratios between 0.15 and 1.56. Cross-linking at a ratio of\n0.15 produced a fluid material of which G ′ could not be probed at 20 °C ( Figure 3 B, light green squares). Upon\nincreasing the ratio of acetylene to azide groups to 0.39, the G ′ of the hydrogels crossed the\nthreshold required for connectivity. When the ratio was increased\nto 1.55, G ′ was lowered again.\nWe conjecture that the modulus decreases at high ratios because when\nacetylene groups are present in excess, a larger fraction of PNIPAM\nmolecules react with just one of their acetylene groups, and the extent\nof interfiber cross-linking is reduced. The cross-linker to fiber\nratio also influences the thermal stiffening of the gels. The storage\nmodulus of the different gels of Figure 3 B was measured as a function of temperature\nduring a linear T -ramp from 20 to 55 °C. The\ndata show that, for networks above the connectivity threshold, the\nnet increase in G ′ resulting\nfrom PNIPAM-AC collapse increases with increasing cross-linker to\nfiber ratios. Specifically, at a 1.55 ratio of acetylene to azide\ngroups, G ′ increases more\nthan 3 orders of magnitude, from 1.5 Pa at 20 °C to 1790 Pa at\n55 °C. Similar trends have also been observed in reconstituted\nactomyosin networks where the magnitude of the stiffening response\nis coupled to the relative amount of force-generating and cross-linking\nproteins. Hence, high [myosin]/[actin] or high [FLNa]/[actin] molar\nratios induce stronger local tension on the filaments, resulting in\nhigher degrees of macroscopic stiffening. 12 , 13 , 34 , 35 To compare\nthe linear storage modulus of PDA/PNIPAM hydrogels to\nthose of a “bare” PDA network lacking a force-generating\nlinker as well as intrafiber cross-links, a direct cross-linking approach\nrecently reported by us was employed ( Figure S6 ). 36 Thus, 15 mg mL –1 PDA/DA-N3 and PDA-DA-AC fiber solutions (each containing 20 mol\n% cross-linkable molecules) were mixed after the covalent fixation\nstep and chemically cross-linked. Since covalent fixation anchors\nthe monomers to the fibers, interfiber migration of reactive groups\nis prevented. Accordingly, all cross-links effectively connect two\ndifferent fibers, and the number of cross-links that contribute to\nthe network’s modulus is maximized. This network was found\nto have a linear storage modulus of 25 Pa, just above the value (9\nPa) of the stiffest network attained using PNIPAM-AC at a [PNIPAM-AC]/[DA-N 3 ] = 0.78 molar ratio, indicating that in the PNIPAM-containing\nnetwork, cross-link density at an optimized cross-linker to fiber\nratio is near the maximum value. Irreversibility of the\nThermally Induced Stiffening Transition To identify the underlying\nmechanisms governing the stiffening\nof PDA/PNIPAM-AC networks, the moduli of the gels were monitored as\nthey were heated to 55 °C and subsequently cooled back to 20\n°C before and after chemical cross-linking of a solution containing\n15 mg mL –1 PDA/DA-N 3 fibers and 7.5 mg\nmL –1 PNIPAM-AC ( Figure 4 A,C). For chemically cross-linked gels ( Figure 4 A,B), heating above\nthe LCST of PNIPAM-AC prompts a 100-fold increase in G ′ that infers strong pulling of PNIPAM-AC on PDA fibers as\nthe linker undergoes a coil-to-globule transition. After cooling back\nto 20 °C, the hydrogel remained in a stiffened state in line\nwith previous observations showing an irreversible fluid–gel\ntransition after removing the gel from the heating source ( Figure S4 ). Interestingly, a solution of PDA\nfibers and PNIPAM-AC coils without covalent connections between the\ntwo components was also found to transition from fluid to gel, featuring\na crossover of G ′ and G ″ at around 35 °C ( Figure 4 D). These results\nsuggest that, much like in composite CNC/PVAc/POEG(M)A networks reported\nby Cudjoe et al., 20 the collapse of the\nthermoresponsive linker brings PDA fibers into physical contact, creating\na percolating network even in the absence of chemical cross-links.\nThe storage modulus of the physical gel after cooling back to room\ntemperature was over 2 orders of magnitude lower than in the cross-linked\nmaterial, indicating that, while the formation of physical connections\nis sufficient to form an elastic material, covalent cross-links strongly\nincrease network connectivity, resulting in much stiffer gels at the\nsame fiber concentration. On cooling back to room temperature, the\nphysical hydrogel relaxes part of the built-up stress as inferred\nfrom a decrease in modulus likely due to PNIPAM chains loosening their\ngrip around the fibers as they swell below their LCST. By contrast,\nin both CNC/PVAc/POEG(M)A composites and biopolymer actin/myosin II\nnetworks, full recovery of the original stiffness is achieved after\ncessation of the active contraction. 13 , 17 , 20 , 31 A possible explanation\nto account for the contrasting irreversibility found in PDA/PNIPAM-AC\nnetworks involves a poorly reversible interaction between the hydrophobic\ncores of two fibers, which are brought together upon heating above\nthe LCST of the PNIPAM component, but do not detach form each other\nwhen the PNIPAN chains swell again upon cooling. Figure 4 Proposed mechanism of\nPDA/PNIPAM-AC stiffening transition. (A)\nMechanism proposed for the stiffening transition at a constant overall\nvolume observed in a covalently cross-linked PDA/PNIPAM-AC network.\n(B) Storage ( G ′ ) and loss\n( G ″ ) moduli vs temperature\nat γ = 1% and ω = 6.28 rad s –1 and a\nheating/cooling rate of 1.25 °C min –1 for a\nnetwork consisting of 15 mg mL –1 PDA (20 mol % DA-N 3 ) cross-linked with PNIPAM-AC using a [PNINAM-AC]/[DA-N 3 ] = 0.78 molar ratio. (C) Sol–gel transition via formation\nof a physical network lacking chemical cross-links between PDA and\nPNIPAM-AC. (D) 15 mg mL –1 PDA mixed with PNIPAM-AC\nusing a [PNINAM-AC]/[DA-N 3 ] = 0.78 molar ratio in the absence\nof added catalyst. Below 30 °C, the data are of limited accuracy,\nas they are dominated by inertia effects with a raw phase angle above\n170° ( Figure S10 ). Mesoscale Structural Characterization of\nthe Hydrogels Relevant insights into the structure of chemically\nand physically\ncross-linked PDA/PNIPAM-AC networks were obtained using small-angle\nX-ray scattering (SAXS). SAXS experiments were performed both below\nand above the LCST of PNIPAM-AC to probe the topology of both systems\n( Figure 5 ). Scattering\nof the physical gel at 20 °C is very similar to the sum of the\nscattering of the separate components, showing that there is little\ninteraction between the two polymers ( Figure 5 A). In the covalent gel at the same temperature,\nthere is excess scattering intensity at low q values\nwith a power law exponent that has increased from −1 to −1.5 (purple\nup triangles in Figure 5 A). This indicates structural heterogeneities at mesoscopic length\nscales (>±15 nm), in line with a calculated mesh size of 82\nnm\nat this concentration ( Table S2 ). Upon\nheating to 55 °C, when the thermosensitive PNIPAM-AC chains collapse\nabove their LCST, the excess forward scattering intensity and power\nlaw exponents increase for both the chemically ( Figure 5 B) and physically ( Figure 5 C) cross-linked hydrogels as the heterogeneities\nherein become more pronounced. In the high q -region,\nthe SAXS profiles recorded above and below the LCST overlay, indicating\nthat the cross-sectional diameter of the PDA fibers in the hydrogels\nremains fixed at approximately 3 nm. Interestingly, a correlation\npeak arises exclusively in the covalently cross-linked hydrogel above\nthe LCST. Fitting with a model to describe aggregation in polymer\nsolutions, 37 which has also been employed\nto describe peptide hydrogels, 38 gives\na correlation length of 1.2 nm, smaller than the diameter of the fibers\n( Figure S8 ). Tentatively, we attribute\nthis feature to the emergence of small domains composed of collapsed\nPNIPAM-AC globules. Figure 5 Small-angle X-ray scattering profiles of un-cross-linked\nphysical\nmixture and covalently cross-linked PDA/PNIPAM-AC (20 mol % DA-N 3 ) gels at 15 mg mL –1 PDA and PNIPAM-AC.\nMolar ratio [PNINAM-AC]/[DA-N 3 ] = 0.78. (A) Comparison\nof the scattering intensities of the physical gel and the covalent\ngel with the sum of the scattering intensities of the separate components.\n(B) Covalently cross-linked gel at 20 and 55 °C. (C) Physical\nmixture at 20 and 55 °C. Nonlinear Mechanics of Prestressed PDA/PNIPAM Hydrogels To more accurately capture the mechanical response of PDA/PNIPAM-AC\nnetworks stiffened via PNIPAM-AC collapse in Figure 3 under externally applied shear stress, a\nbenchmarked rheological prestress protocol was applied. 39 Accordingly, the differential modulus K (the elastic part of which relates the change in stress\nwith strain, K ′ = δσ/δγ)\nwas measured by parallel superposition of an oscillatory and a steady\nprestress σ. The stiffness—quantified by K′ —of most synthetic hydrogels based on flexible polymer chains\nis constant at biologically relevant stresses. 21 By contrast, gels reconstituted from most intra- and extracellular\nfilamentous proteins are known to exhibit two distinct regimes: a\nlow-stress linear regime, where K ′ is equal\nto the plateau storage modulus G 0 , and\na high-stress nonlinear regime, where K ′ increases\nwith σ as K ′ ∝ σ m , with m being the so-called stiffening\nexponent. K ′ vs σ data were recorded\nafter applying a T -ramp from 20 to 55 °C (in Figure 3 ) and cooling back\nto 20 °C. Since the stiffening transition for covalently cross-linked\nnetworks was almost fully irreversible, the moduli of the gels at\n20 °C were nearly identical to the values at 55 °C. Thus,\nstiffened PDA/PNIPAM networks from Figure 2 B were subjected to a range of steady prestress\nσ at 20 °C. All networks exhibited an apparently linear\nresponse over a concentration-dependent range of applied σ ( Figure 6 A). However, at a\ncharacteristic critical stress (σ c ) the moduli of\nthe gels begins to increase. Strikingly, a 4-fold increase in PDA\nconcentration at a [PNIPAM-AC]/[PDA] ratio of 0.78 combined with an\nexternally applied stress raises the plateau storage modulus\nfrom 2 Pa to 10 kPa, nearly 4 orders of magnitude. Concomitantly,\nthe stress at failure (σ max ) increases with concentration\nby 3 full decades. For the highest PDA concentration, σ max reaches a value of 1.3 kPa, vastly surpassing the maximum\nstress of synthetic polyisocyanopeptides (PICs)-based biomimetic gels,\ni.e., σ max ≈ 40–100 Pa, where the solubility\nrange of PIC polymers is considerably narrower. 40 , 41 To quantify the dependence on c of both G 0 and σ c , scaling analysis\nwas performed and revealed a G 0 ∝ c 5.1 and G 0 ∝\nσ c 5.1 relationship over the whole c range studied ( Figure S9 ).\nSuch scaling differs from the typical square dependence found in both\ntheoretical and literature values. 7 , 40 , 42 , 43 We hypothesize that\nthe principal effect causing the strong concentration dependence of G 0 is, among other factors, the concentration-dependent\nratio of intrafiber to interfiber cross-linking. Additionally, we\nwarn against overinterpretation, as we do not have scaling data over\neven a single decade and therefore cannot rule out that the observed\ndependence is a crossover effect. Figure 6 Nonlinear mechanical response of PDA/PNIPAM-AC\nhydrogels to externally\napplied shear stress after heating the gels to 55 °C and cooling\nback to 20 °C. (A) Differential modulus K ′\nplotted against stress σ for different concentrations of PDA\ncross-linked using a fixed molar ratio [PNINAM-AC]/[DA-N 3 ] = 0.78 obtained from prestressed gels of Figure 3 A. (B) Plot of K ′\nvs stress σ with K ′ normalized to G 0 and σ normalized to σ c , showing collapse onto a single master curve with K ′ ∝ σ 1 at high σ. (C) K ′ vs σ measured at 20 °C for 15 mg mL –1 PDA cross-linked using varying [PNINAM-AC]/[DA-N 3 ] molar ratios obtained from prestressed gels of Figure 3 B. (D) Plot of K ′ vs stress σ with K ′\nnormalized to G 0 and σ normalized\nto σ c , showing collapse onto a single master curve.\n(E) Stiffening factor at failure K ′ max / G 0 plotted against [PNINAM-AC]/[DA-N 3 ] molar ratio. (F) Differential modulus K ′ plotted against stress σ for a PDA hydrogel (12 mg\nmL –1 ) cross-linked using a [PNINAM-AC]/[DA-N 3 ] = 0.78 molar ratio, measured at different temperatures.\nThe gel was brought to its σ max only in the last\nrun at 55 °C. The nonlinear mechanics\nof PDA/PNIPAM hydrogels were compared to\nthose of the “bare” PDA reference network lacking a\nthermoresponsive linker as well as intrafiber cross-links ( Figure S6A ). Such networks, obtained by cross-linking\nfibers with either 20 mol % DA-AC or DA-N 3 analogues showed\nidentical stiffening behavior to the PDA/PNIPAM hydrogels ( Figure 6 A; red triangles),\nindicating that the nonlinear elastic response of these materials\nis exclusively governed by the semiflexible matrix of PDA fibers resisting\nbending and elongation. All curves in Figure 6 A, including the reference curve of the bare\nPDA network, were reduced\nto a single master curve by normalizing K ′\nto its value in the low-stress linear regime G 0 and by normalizing σ to σ c ( Figure 6 B). The master curve\nexhibits power-law dependence K ′ ∝\nσ 1 above σ c . This value of the stiffening\nexponent ( m ) features universally in biopolymer materials\nat all length scales. In subcellular scales, m =\n1 has been reported for reconstituted, active networks of FLNa-cross-linked\nactin stiffened by myosin II. 13 At whole-cell\nscales, m = 1 is robustly seen in entire fibroblasts. 44 Macroscopically, m = 1 is likewise\nreported for extracellular hydrogels of reconstituted type I collagen. 45 This match between PDA/PNIPAM-AC hydrogels and\nfilamentous biomaterials highlights the biomimetic nature of these\nmaterials. To assess the effect of [PNINAM-AC]/[DA-N 3 ] ratio on\nthe nonlinear mechanics of the hydrogels, the same prestress protocol\nat 20 °C was applied to gels of Figure 3 B. Figure 6 C–E clearly show that increasing the ratio of\nPNIPAM-AC to DA-N 3 at a fixed concentration of PDA (15\nmg mL –1 ) extends the range of nonlinear deformation,\nresulting in a concomitant increase of the stiffening factor from\n3.5 at a ratio of [PNINAM-AC]/[DA-N 3 ] = 0.15 up to 7.7\nat [PNINAM-AC]/[DA-N 3 ] = 1.5 (in Figure 6 E). This supports the notion that at higher\nPNIPAM to fiber ratios enhanced intrafiber cross-linking reinforces\nthe fibers and extends the range of nonlinear deformation, as has\nrecently been reported by us by making use of multiarm cross-linkers. 41 The nonlinear mechanics of physical networks\nwere also studied\nin comparison to their covalently cross-linked counterparts. Thus,\nhydrogels from Figure 4 B,D were subjected to a range of steady prestress after heating to\n55 °C and cooling back to 20 °C ( Figure S11 ). Strikingly, the hydrophobic connections holding the physical\ngel together were strong enough to support a regime of nonlinear deformation\nprior to failure. However, both plateau modulus and stiffening factor\nwere substantially lower than in the cross-linked material owing to\na lower network connectivity and weaker connections between fibers,\nrespectively. Having shown that PDA fibers cross-linked with\nthermoresponsive\nPNIPAM-AC exhibit thermo- and mechanoresponsiveness, we set out to\nstudy the magnitude of the response of PDA/PNIPAM gels to combined\nmechanical and thermal stimuli. To this end, we carefully subjected\na sample of the hydrogel to a range of applied stresses below σ max while continuously recording K ′\nat different temperatures, both below and above the LCST of PNIPAM-AC. Figure 6 F shows that the\ncombination of externally applied shear stress and internally generated\ncontractile forces through PNIPAM-AC collapse triggers a strong response\nthat drives the network from an initial soft state with an associated\nmodulus of 1.5 Pa at 20 °C to a final modulus of 2000 Pa at 55\n°C just before failure."
} | 7,918 |
36278719 | PMC9624335 | pmc | 6,073 | {
"abstract": "Antifogging surfaces with unique properties to migrate severe fog formation have gained extensive interest, which is of particular interest for transparent substrates to obtain high visibility and transparency. To date, a large number of strategies including superhydrophilic or superhydrophobic surfaces and titanium dioxide (TiO 2 )-based composite coatings have been developed based on different mechanisms. Although these surfaces exhibit effective antifogging properties, the rigid nanostructures, cumbersome preparation, and the need for UV light excitation largely limit their widespread applications. Herein, we report a zwitter-wettable antifogging and frost-resisting coating through a fast UV-curable cross-linking of copolymer with benzophenone groups. A series of random copolymers consisting of hydrophilic hydroxyethyl methacrylate (HEA), hydrophobic methyl methacrylate (MMA), and benzophenone-based acrylate units are developed by thermally triggered free-radical polymerization. Upon UV light irradiation, a highly efficient antifogging/frost-resisting coating is covalently bonded on a polycarbonate plate surface, maintaining a light transmission higher than 85%, which was confirmed in both high and low temperature anti-fog tests. Moreover, the wetting behaviors reveal that the antifogging performance exhibited by the zwitter-wettable surface mainly relies on its surface water-adsorbing capability to imbibe condensed water vapor on the surface outmost layer. Notably, the antifogging/frost-resisting behaviors can be well regulated by adjusting the hydrophilic/hydrophobic units, due to the proper balance between the water-adsorption and coating stability. Owing to its simplicity, low-cost preparation and high efficiency, this UV-curable acrylate antifogging coating may find a wide range of applications in various display devices in analytical and detection instruments.",
"conclusion": "4. Conclusions We have successfully developed a zwitter-wettable antifogging and frost-resisting coatings, through a UV-curable copolymer consisting of HEA, MMA, and 4-BP acrylate. Owing to the introduced BP groups in the copolymer chains, the copolymers can be easily bonded on the PC substrate surface upon a convenient UV light irradiation. Because of the suitable hydrophilic-hydrophobic balance, the C-70% was considered as the optimized sample, exhibiting both remarkably high antifogging and frost-resisting performances, with more than 85% light transmission. The antifogging mechanism beneath the coating as revealed by its time-dependent wetting behavior mainly relied on its surface water-adsorbing capability to imbibe condensed water vapor on the surface outmost layer, hence avoiding the surface fog formation. In addition, the stability of the coating was studied by exposing the coating to UV radiation, water immersion and heat treatment, and the results showed that the treated coatings still maintained good antifogging properties. Benefiting from the merits of simplicity, low-cost preparation, and high efficiency of the coating preparation, we believe that this UV-curable acrylate antifogging coating may find a wide range of applications in various display devices, ranging from goggles to medical detection devices.",
"introduction": "1. Introduction Fogging and frosting are prevalent in nature and cause a lot of inconvenience to humans daily [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. Owing to the rapid changes in temperature and humidity, saturated water vapor in the air condenses and forms fog droplets on the solid substrates [ 11 ]. The formation of fog droplets will not only cause surface wetting, but also has a great impact on the light transmission of the transparent materials, resulting in a significant reduction in their view clarity [ 12 , 13 ]. For example, the presence of a fog layer can largely reduce the solar energy conversion rate of solar panels [ 14 , 15 , 16 ]. Severe fog formation can blur the vision of vehicle drivers, which can easily cause traffic accidents [ 17 ]. In addition, in the field of medical testing, fogging of the testing lens during surgery can even cause catastrophic medical accidents [ 9 , 18 ]. So far, a large number of antifogging strategies have been designed and prepared to mitigate severe fogging and frosting. Among those, superhydrophilic surfaces that can get extremely low water contact angles within 0.5 s or less, have the ability to significantly reduce light scattering by allowing water to spread into a thin film [ 19 , 20 ]. For conventional superhydrophilic surfaces, both high surface energy and suitable roughness scales are extremely necessary to enhance the surface super-wetting behaviors [ 21 ]. Till now, various techniques such as plasma etching [ 22 ], layer-by-Layer (LbL) [ 23 ], templating method [ 24 ] have been adopted to develop the superhydrophilic antifogging surfaces. However, those techniques generally require either complicated procedures or special instruments to get the appropriate surface roughness. Additionally, the superhydrophilic surfaces can also be prepared by introducing the photochemically active materials (e.g., TiO 2 ) into its coating [ 25 , 26 ]. However, most of those coatings must be exposed under UV illumination to obtain the superhydrophilic anti-fogging properties. On the other hand, some superhydrophobic surfaces also displayed antifogging behaviors due to its super-repellency against water droplets [ 27 , 28 , 29 ]. Considering the micro-scale fog droplets formed on the surface, only some special superhydrophobic surfaces with precisely controlled roughness and topography can exhibit the qualified antifogging ability. Typical superhydrophobic surfaces are opaque and have very low light transmission, not to mention that the micro and nano structures are easily damaged and not easy to handle, all of which lead to difficulties in the application of the anti-fogging properties of superhydrophobic surfaces. Recently, some coatings with zwitter-wettability have been reported to have good anti-fogging behavior [ 30 , 31 , 32 , 33 , 34 , 35 ]. Unlike the previously reported superhydrophilic or superhydrophobic surfaces, these zwitter-wettable coatings have moderate water absorption capacity. The anti-fogging mechanism of the coatings is believed to be that their coatings can strongly adsorb water vapor to their bulk materials rather than forming condensed water droplets on the surfaces, finally resulting in a totally clear coating surface. Some zwitterionic wettable coatings were prepared by grafting with oligomers consisting of perfluoroalkyl and polyethylene glycol (PEG) segments [ 36 ] or by a layer-by-layer (LbL) assembly method based on chitosan/Nafion systems [ 37 ]. More recently, some polymeric coatings with a semi-interpenetrating polymer network (SIPN) have been developed, through a binary or terpolymer acrylic polymers. Moreover, some zwitter-wettable coatings with both antifogging/antibacterial performances can also be developed by the combination of a cationic copolymer and a hydrophilic copolymer [ 38 , 39 ]. Although good anti-fog properties have been obtained for these coatings, there is still a strong demand for preparing these wettable coatings by a more rapid and convenient method. Although the coatings exhibit effective antifogging performances because of the water-adsorbing behaviors, the inhaled water may turn into ice crystal under an extremely cold condition below the water freezing point, which inevitably decrease the light transmission. Nature always offers immense inspirations for creating functional material and surfaces. The roots of some overwintering plants, with high water content, can tolerate extremely cold temperatures. The key to this property is that the water present in the roots is in a nonfreezing or intermediate water states, thus avoiding the formation of ice crystal as well as the damage of plant cells [ 40 , 41 , 42 ]. Inspired by this concept, we herein develop a zwitter-wettable acrylate coating with antifogging and frost-resisting performances through a facile UV-curable cross-linking of copolymer with benzophenone groups. Initially, a series of copolymers consisting of hydrophilic hydroxyethyl methacrylate (HEA), hydrophobic methyl methacrylate (MMA), and benzophenone-based acrylate (BP-Acrylate) units were synthesized by thermally triggered radical polymerization. The hydrophilic-hydrophobic balance of the copolymers can be well adjusted by adjusting the molar ratio of HEA/MMA. The structure and molecular weight of the prepared copolymers were investigated by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). Subsequently, the copolymers were coated on the surface of PC plates to obtain covalently bonded anti-fogging coatings under UV light. The anti-fog and anti-frost properties were investigated, and the antifogging mechanism was studied by surface wetting behavior.",
"discussion": "3. Results and Discussion 3.1. Preparation and Properties of the Zwitter-Wettable Coatings The zwitter-wettable coating was successfully prepared according to the following procedure. First, the copolymers were synthesized by a thermally triggered free-radical polymerization reaction with the hydrophilic HEA, hydrophobic MMA and 4-BP-acrylate units. Herein, the 4-BP-acrylate groups were served as UV photo-initiator to immobilize the copolymer on the PC substrate surface. The mechanism of the UV triggered surface grafting is that the BP-based groups were excited under UV irradiation to a singlet state and jump to a triplet state which then underwent hydrogen-abstracting reaction from substrates while the resulting BP radicals tended to participate in coupling reaction to covalently bond the copolymers to the substrate surface [ 43 ]. By adjusting the mass percentages of the HEA/MMA, the surface zwitter-wettable behaviors can be well controlled. A great number of hydrophilic HEA units in the copolymer backbone could enhance its water adsorption capability, hence improving its antifogging performance. The Mn of the copolymers for P-30%, P-50%, P-70%, P-90% were 79.1 KDa, 71.3 KDa, 69.2 KDa, and 63.4 KDa, respectively, as determined form the GPC test. The chemical structures of the copolymer were investigated by H-NMR analysis ( Figure 1 a). The methylene (CO-CH 2 -CH 2 ) resonance from polyHEA segment appears at 3.9 ppm. The other methylene (−CH 2 -CH 2 -OH) and the methyl protons from poly(MMA) appeared at around 3.6 ppm. Moreover, multiple proton peaks at around 6.8, 7.6, 7.8 ppm were attributed to the benzophenone groups. Taken together, those 1H NMR results confirmed the successful preparation of the copolymers. Additionally, the molar ratios of HEA/MMA/4-BP acrylate in copolymers were also roughly estimated by the peak intergration of the 1 H NMR spectra which were consistent with the initial feed ratio by conversion to mass fractions as mentioned before. After being treated under UV light irradiation and sufficient cleaning to eliminate the unbonded copolymers, the resulting coatings (C-30%, C-50%, C-70%, and C-90%) were evaluated by ATR-FTIR spectroscopy to check the chemical composition of the surfaces ( Figure 1 b). The broad band at 3540 cm −1 stemming from the −OH stretching showed obvious enhancement due to increased HEA contents in the copolymers. The C = O stretching bands in HEA, MMA, and 4-BP acrylate were also observed at ~1730cm −1 , indicating the presence of the copolymers on the substrate surfaces. 3.2. Antifogging Performances Before antifogging test, the light transmission values of the coatings (C-30%, C-50%, C-70%, and C-90%) were evaluated with the control PC plate as the reference, as displayed in Figure 2 a. All the coatings demonstrated high light transmission values (more than 91%), which were comparable to that of the bare PC plate (~92%). The results demonstrated that the introduced coatings on the surface possessed good light transparency, the coating material itself does not cause any significant light absorption. However, just through a simple breath fogging experiment, a clear difference in the antifogging performance of the sample surface can be found. Compared to the blurred picture observed by the control lens, the surfaces treated by antifogging coatings for both the goggles and glasses showed clear views behind the lens ( Figure 2 b). As one of the most prevalent natural phenomena, fog droplets can form on a variety of surfaces under the right conditions, which can trigger severe light reflection and refraction and reduce the light transmission of transparent substrates. Antifogging surfaces are often considered to be one of the most promising strategies for mitigating fog formation, but their performances can be significantly affected by different fogging environments. In this section, antifogging tests in either hot (80 °C) fog conditions were first conducted, and the related antifogging performances were qualitatively and quantitatively recorded. As for the antifogging test, all the coated PC films were placed above 5 cm over the hot water (80 °C) for 60 s with the coated side face down, the fogging images were recorded with a digital camera and the light transmission were examined by UV-vis spectrophotometer immediately ( Figure 3 a,b). For the control PC plates, a heavy fog layer rapidly appeared on the downside surface upon exposure to water vapor. Prolonging the exposure time to 60 s made these situation even worse, larger fog droplets stemming from the vapor condensation gradually appeared on the film surface, finally leading to a completely non-transparent film. The fog layer formed caused the background image blurred. When the exposure time was extended to 60 s, dense droplets of fog gradually appeared on the film surface, finally resulting in a completely opaque film. For comparison, the coated samples exhibited quite different antifogging behaviors. The C-30% and C-50% samples showed good anti-fogging performance in the initial period of 5–6 s. As the incubation time was prolonged, a tendency of fog droplet formation and becoming larger began to appear on the surface. When prolonging the incubation time to 60 s, non-continuous hydration layers were formed on both two C-30% and C-50% surfaces. In contrast, both the C-70% and C-90% displayed remarkably enhanced antifogging performances in hot and humid environments. The antifogging properties of the sample surfaces were also evaluated quantitatively, by immediately collecting the light transmission over the range of 400–700 nm. The overall transmittance results are generally consistent with the anti-fog images, with all samples exhibiting very different light transmissions. Among these, the control PC plates exhibited relatively low transmittance values (~34%) in the 400–800 range, compared to an initial value of ~92% prior to the fogging test. For the coated samples, the antifogging performances of the samples were closely related to the contents of hydrophilic monomers in the copolymers, with a progressive increase in light transmission from 52% to 61% for C-30% and C-50% accompanied by an increase in HEA content. Previous reports have confirmed that the presence of hydrophilic components in the coating can effectively absorb the fog droplets formed on the surface [ 32 ]. More hydrophilic components in the coating will result in a stronger water absorption capability, which also enhances the surface antifogging performance. When the HEA mass percentage was increased to 70%, the C-70% coating achieved ~90% light transmission even after fogging test. However, by continually increasing the HEA mass content to 90%, the light transmission of the C-90% showed a slight decrease in light transmission. This phenomenon may be due to the excessive content of hydrophilic component in the coating, which absorbs excessive water vapor and causes excessive swelling of the coating, finally resulting in a reduction of its surface light transmission. Therefore, C-70% proved to be the optimal sample to get the superior antifogging effect against hot moist air. 3.3. Frost-Resisting Performances Besides the hot temperature antifogging performance, the frost-resisting property is also critical for a functional surface to get more broad antifogging applications. When water vapor in a humid environment comes into contact with a cold surface, the water vapor at the low temperature material interface becomes supersaturated and quickly condenses on the surface to form fog droplets first and subsequently turns into a frost layer due to the extremely low surface temperature. Since this type of frost layer is formed directly from condensed water droplets on the surface, it has a severe reflective and interfering behavior toward light, which greatly reduces the transmittance of visible light. To challenge a harsh fogging situation, the samples were stored in a refrigerator at −20 °C for 2 h and then exposed to the ambient condition (∼20 °C, 35–40% relative humidity) ( Figure 4 a,b). When the control PC plates were transferred from an extremely cold environment to an ambient environment, a fog layer was formed on the surface immediately, which was then converted into a frost layer due to the cold temperature of the substrate. Both the fog and frost layers formed on the surface have serious impacts on the light transmission of the sample, and only a very blurred image can be observed through the control PC plate. In comparison, the coated samples showed varied surface frost-resisting performances. The C-30% did not display substantial frost-resisting performance and the background image was very unclear. In contrast, the C-50% showed an obviously enhanced frost-resisting behavior, while the C-70% and C-90% samples both displayed significantly enhanced frost-resisting performances, with no fog or frost layer present on the surface during the whole frosting test, although the C-70% sample showed even superior clarity than that of the C-90%. The light transmission confirmed that the frost-resisting performances of the samples were closely related to the hydrophilic HEA contents, both the C-70% and C-90% showed higher light transmissions than 88%. Similar to the antifogging results mentioned above, the coatings with excessive HEA will weaken the frost resistance. Previous studies have confirmed that excessive hydrophilic content in copolymer can lead to excessive water uptake by the coatings, which can produce inhomogeneous water domains during the frost formation at low temperatures, thus remarkably reducing the light transmission to certain extent [ 30 ]. 3.4. Surface Wetting Behaviors The wetting properties of the sample surfaces have a significant effect on their anti-fogging performances. Among them, the superhydrophilic surface is used to achieve the anti-fogging performance by forming a smooth and uniform hydration thin layer by rapidly spreading fog droplets on the surface, while the superhydrophobic surfaces repel the micro-sized fog droplets from the surface by using the unique super-repellence of the surface. To investigate the anti-fogging performance of the amphiphilic hydrophilic surface, the wettability of the sample surface with time was also investigated. To understand the anti-fogging mechanism of the zwitter-wettable coating surface, the time depended water CA values was monitored, and the changes of the CA values as well as the diameter changes of water droplet on the coating surface were recorded in each 4 s interval within 80 s incubation period ( Figure 5 ). As shown in Figure 5 a, the control PC plate showed relatively high CA values ~88°, and the CA values only showed a slight decrease during the incubation time of 80 s. The result confirmed that the saturated water vapor tends to generate tiny droplets on the hydrophobic surface when it encounters a temperature change. The coated surface also exhibited relatively high initial contact angles, with CA values of ~70°, 65°, 53°, and 49° for C-30%, C-50%, C-70%, and C-90%, respectively, which is quite different from typical superhydrophilic antifogging surfaces with very low water CA (≤5°). These results confirm that an antifogging surface does not need to be superhydrophilic to diffuse condensed droplets into a thin water layer. It was noteworthy that the water CA value of the coated surface varied with time and decreases significantly during the incubation time. The CA of the water droplets on the bare PC plates surface decreased by only 3° during the 80 s incubation time of the water droplet on the surface. In contrast, the CA of the C-30% dropped significantly, from an initial 65° to 50°, with a CA decrease of ~15°. The continuous increase in the content of hydrophilic HEA units resulted in more pronounced decreases in CA values. More significant decreases in CA values, ~20°, 29°, and 30° were observed on the C-50%, C-70% and C-90% surfaces, respectively. These results indicated that the higher content of HEA units in the copolymer could result in a stronger water absorption capacity, which was closely related to the antifogging properties of the surface. In addition, the variation of water droplet diameter over time on the coated surface was also evaluated ( Figure 5 b). The coated surfaces with more HEA content exhibited a more pronounced increase in wetting diameter compared to the bare PC surface. Among them, the C-30% and C-50% samples showed ~22% and 24% increase in wetting diameter. Further increase in HEA content led to a continuous increase in wetting diameter, with ~37% and ~43% increase found on the C-70% and C-90% surfaces. This phenomenon further confirmed the fact that the hydrophilic HEA segment in the coating facilitates the adsorption of water, which leads to an expansion of the water wetting area. Together with the anti-fogging results, we can get the conclusion that this zwitterionic wettable coatings with a suitable hydrophilic-hydrophobic balance possess a suitable hydrophilic-hydrophobic balance and thus have excellent antifogging properties. 3.5. Surface Stability The surface properties of the coating may be affected when exposed to some harsh environments (e.g., UV irradiation, water immersion, heat treatment), which in turn can lead to a weakening or even loss of surface antifogging functions and seriously affect the lifetime of the functional surface. To verify whether the resulting samples were affected by the above treatments, the C-70% samples were subjected to UV irradiation, water immersion, and heat treatment, respectively, and their surface antifogging properties were quantitatively studied, according to the above-mentioned antifogging test ( Figure 6 ). After 24 h UV irradiation (0.8 mW cm −2 , 360 nm), the C-70% maintained more than 90% light transmission. The following fogging test confirmed that the antifogging performance of the UV-irradiated samples was slightly reduced compared to its initial antifogging behavior (~90%) of the samples without UV irradiation, but still maintained a high light transmission rate of more than 87%. For those samples immersed in water bath for 24 h, and the surface still maintained relatively high light transmittance (~89%), and also possessed with highly efficient antifogging performance. Moreover, the samples also exhibited excellent thermal stability after being heated in an oven at 100 °C for 24 h, with ~88% light transmission after fogging test. Together, the above results demonstrated that the prepared samples have excellent coating stability and can maintain significant antifogging properties even after being treated by UV irradiation, water immersion, and heating treatment."
} | 5,925 |
21406598 | PMC3055163 | pmc | 6,075 | {
"abstract": "Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal gene transfer is often mediated by conjugative genetic elements that transfer directly from cell to cell. Integrative and conjugative elements (ICEs; also known as conjugative transposons) are mobile genetic elements that reside within a host genome but can excise to form a circle and transfer by conjugation to recipient cells. ICEs contribute to the spread of genes involved in pathogenesis, symbiosis, metabolism, and antibiotic resistance. Despite its importance, little is known about the mechanisms of conjugation in Gram-positive bacteria or how quickly or frequently transconjugants become donors. We visualized the transfer of the integrative and conjugative element ICE Bs1 from a Bacillus subtilis donor to recipient cells in real time using fluorescence microscopy. We found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, ICE Bs1 spread rapidly from cell to cell within the chain by additional sequential conjugation events. This intrachain conjugation is inherently more efficient than conjugation that is due to chance encounters between individual cells. Many bacterial species, including pathogenic, commensal, symbiotic, and nitrogen-fixing organisms, harbor ICEs and grow in chains, often as parts of microbial communities. It is likely that efficient intrachain spreading is a general feature of conjugative DNA transfer and serves to amplify the number of cells that acquire conjugative mobile genetic elements.",
"discussion": "DISCUSSION We used two different methods to visualize conjugative DNA transfer between donor and recipient cells. In one case, we visualized the DNA that was transferred from cell to cell. In the second, we used conditional protein degradation to identify cells that acquired the horizontally transferred element. We found that successful conjugation of the integrative and conjugative element of B. subtilis , ICE Bs1 , occurred with no obvious orientation of the donor and recipient. That is, transfer of DNA from a donor into a recipient appeared to occur at a cell pole or along the lateral cell surface. Furthermore, when acquired by a cell in a chain of cells, ICE Bs1 spread rapidly to other cells in the chain through sequential transfer events as transconjugants quickly became donors. Integration and stable maintenance of ICE Bs1 in the host chromosome requires repression of ICE Bs1 gene expression from the rightward promoter P xis ( Fig. 1 ). Derepression of P xis leads to expression of genes needed for ICE Bs1 excision and conjugation ( 11 , 15 ). The excised circular form of ICE Bs1 is required for its dissemination to recipients. Our results indicate that soon after receiving ICE Bs1 , a very high percentage of transconjugants become donors by expressing conjugation genes. The ability of a transconjugant to become a donor is likely influenced by the kinetics of repression of P xis , which in turn is influenced by the kinetics of accumulation of the ICE Bs1 repressor ImmR. ImmR both activates and represses its own expression, creating a homeostatic autoregulatory loop ( 15 ). Initially, there is no ImmR in a newly formed transconjugant, permitting transcription from P xis and expression of ICE Bs1 conjugation genes. However, in the absence of an inducing signal, expression and accumulation of ImmR in the transconjugant will eventually repress P xis , allowing integration of ICE Bs1 into the chromosome. This type of regulatory circuit is common in mobile genetic elements, notably in bacteriophage ( 30 , 31 ), and is important in fate determination for such elements. In ICE Bs1 , this circuit likely allows switching between an active dissemination mode (excision and gene expression) and a quiescent inactive mode (integration and repression). Our studies indicate that a delay in ICE Bs1 integration and transcriptional repression in transconjugants contributes to the spread of ICE Bs1 in cell populations. Much is known about conjugation and conjugative elements of both Gram-negative and -positive bacteria ( 4 , 32 , 33 ). In most cases, transfer efficiencies of a few percent are considered high. Our results indicate that conjugation efficiencies in cell chains can be >50%. A different mechanism for efficient dispersal of a mobile element has been described for Streptomyces ICEs that can exist as stable plasmids. Plasmid spreading through Streptomyces mycelia depends on spreading proteins (Spd) and is independent of conjugation proteins (summarized in references 32 and 34). In contrast, transfer of ICE Bs1 to cells in a chain requires the conjugation machinery and is not due to replication and segregation of the plasmid form of ICE Bs1 . Many bacterial species, including pathogenic, commensal, symbiotic, and nitrogen-fixing organisms, grow in chains and harbor conjugative elements. In addition, microbial biofilms are often composed of long chains or aggregates of connected cells ( 17 ). It seems likely that efficient intrachain spreading is a general feature of conjugative DNA transfer and probably serves to rapidly amplify the number of cells that acquire conjugative mobile genetic elements. When cells are present in a chain, they are in intimate contact with other cells in a pole-to-pole configuration. The high efficiency of intrachain conjugation is likely due to close and stable cell-cell contact. The high concentration of conjugation proteins at donor cell poles ( 21 – 24 ) might also contribute to the efficient pole-to-pole transfer in cell chains."
} | 1,436 |
36652484 | PMC9942920 | pmc | 6,076 | {
"abstract": "Significance The long-term impacts of infection on the microbiota and its regulation of host physiology are poorly understood. Here, we report that long-term alterations to the gut microbiota following a single, acute episode of bacterial or protozoan gut infection can remodel host metabolism to preferentially and more efficiently consume carbohydrates. This infection-triggered metabolic remodeling ultimately results in white adipose tissue expansion and host weight gain. Furthermore, in the context of low limited nutrient availability, infection-triggered carbohydrate metabolism benefits host fitness by preventing host stunting. Our study suggests a new perspective in which infection (pathogen-induced stress) can be co-opted as a cue to prime host adaptation to nutritional stress.",
"conclusion": "Conclusion Together, our work highlights the fundamental role of infection in mediating host adaptation to nutrient precarity. By probing the long-term impacts of infection on host metabolism, we discovered that infection-induced gut microbiota optimizes host metabolism toward the usage of carbohydrates. Because carbohydrates are often a more available nutrient source than protein or fat, we speculate that infection-optimized carbohydrate metabolism may be adaptive in under-resourced settings where infection and nutrient precarity often co-occur. In contrast, in settings where carbohydrates are overly accessible (such as with a high-sugar Western diet) or restricted (ketogenic diet), infection-induced carbohydrate metabolism may instead be maladaptive.",
"discussion": "Discussion The long-term consequences of environmental stressors such as infection on host physiology remain largely unexplored. Here, we show that infection-elicited gut microbiota can remodel host metabolism to preferentially utilize carbohydrates, resulting in heightened glucose disposal, WAT expansion, and weight gain. Furthermore, infection-optimized carbohydrate metabolism could sustain host fitness in the context of limited protein and fat availability, preventing the growth stunting (malnutrition) that otherwise occurs in infection-naïve mice. Our observations add to a growing body of evidence that environmental stressors are in fact necessary for the full development and optimization of host physiology ( 18 , 36 ). For example, we previously uncovered that nutrient restriction can enhance the formation of immunological memory and thus resistance to subsequent infection ( 36 ), while in the present work, we identify essentially the reverse phenomenon that prior infection can trigger host adaptation to nutrient restriction. This reciprocal regulatory structure (i.e., infection priming for nutrient restriction, and nutrient restriction priming for infection) is likely adaptive and may have arisen as a defense against the frequent co-occurrence of food precarity with infection and other stressors throughout mammalian evolution ( 31 , 33 , 37 ). In modern human settings, food precarity primarily occurs among under-resourced populations, where the staple foods (e.g., corn, potato, and rice) are often high in carbohydrates ( 38 ). Because of this, food precarity can preferentially restrict access to protein and fat. Such skews in nutrient intake, through diets high in carbohydrates but low in protein and fat, potentially contribute to the high rates of malnutrition among under-resourced populations ( 39 ). This widespread condition induces a range of developmental defects, such as growth stunting ( 7 ), and it often afflicts regions simultaneously burdened with high rates of infectious disease ( 31 ). Our finding that infection can promote resistance to malnutrition, particularly malnutrition induced by limited access to protein and fat, raises the intriguing possibility that this phenomenon may help to support human fitness and survival in under-resourced settings. Though we firmly established a role for the gut microbiota in our observed phenotypes, we presently lack a mechanistic understanding of how infection-elicited microbiota can remodel distal tissues (i.e., WAT) and systemic physiology (i.e., carbohydrate metabolism). We hypothesize a prominent role for microbiota-associated molecular patterns (MAMPs) and/or metabolites, since these molecules are known to mediate much of the microbiota’s control over host metabolism, for example by modulating the activity of enteroendocrine cells ( 40 ). These specialized gut epithelial cells secrete systemically acting hormones that stimulate production of the glucose-disposing signal insulin ( 41 ). Furthermore, enteroendocrine hormones can regulate distal tissues such as WAT ( 42 , 43 ), potentially leading to WAT expansion and weight gain ( 43 , 44 ). Supporting a role for microbiota-derived MAMPs/metabolites in modulating the enteroendocrine cell activity of previously infected mice, Parasutterella , which we showed are elicited by prior infection and sufficient to enhance glucose homeostasis, have been shown to directly adhere to gut epithelial cells ( 45 ) and to localize along the entire length of the gastrointestinal tract, both the large intestine and the upper ( 46 ) and lower ( 45 ) small intestine (duodenum and ileum). These two sites are, respectively, where two major types of enteroendocrine cells, K and L cells, are primarily found ( 47 ). Furthermore, Parasutterella produce the MAMP lipopolysaccharide ( 45 ) and enhance host production of bile acid metabolites ( 46 ), both of which have been shown to modulate enteroendocrine cell activity ( 48 , 49 ). In ongoing studies, we are exploring how Parasutterella -associated MAMPs/metabolites potentially synergize to enhance host metabolism long-term after infection. Study Limitations. Extrapolation of our results to humans is limited by the fact that our study took place in mice and was highly controlled (used defined pathogens and diets). Further studies of heterogeneous human cohorts are warranted to assess the impact of prior infection and/or other environmental exposures on host metabolic status later in life."
} | 1,516 |
38834547 | PMC11150272 | pmc | 6,077 | {
"abstract": "Liquid-solid contact electrification (CE) is essential to diverse applications. Exploiting its full implementation requires an in-depth understanding and fine-grained control of charge carriers (electrons and/or ions) during CE. Here, we decouple the electrons and ions during liquid-solid CE by designing binary superhydrophobic surfaces that eliminate liquid and ion residues on the surfaces and simultaneously enable us to regulate surface properties, namely work function, to control electron transfers. We find the existence of a linear relationship between the work function of superhydrophobic surfaces and the as-generated charges in liquids, implying that liquid-solid CE arises from electron transfer due to the work function difference between two contacting surfaces. We also rule out the possibility of ion transfer during CE occurring on superhydrophobic surfaces by proving the absence of ions on superhydrophobic surfaces after contact with ion-enriched acidic, alkaline, and salt liquids. Our findings stand in contrast to existing liquid-solid CE studies, and the new insights learned offer the potential to explore more applications.",
"introduction": "Introduction Contact electrification is an interfacial process whereby static charges are generated during the contact and separation of two surfaces 1 . CE ubiquitously occurs at various interfaces, particularly at the solid-solid and liquid-solid interfaces. Solid-solid CE has experienced extensive research, and three types of charge carriers 2 , including electrons 3 , ions 4 – 6 , and materials 7 , 8 , have been used to account for the charge generation during CE between different types of solid surfaces. In comparison, liquid-solid CE is still not well understood despite its significance in various applications, such as water energy harvesting, microfluidics, interfacial chemistry, surface wet cleaning, etc 9 – 14 . The main bottlenecks hitherto in understanding the mechanism of liquid-solid CE lie in ascertaining charge carriers, which may involve ions, electrons, or both 15 – 24 . Another challenge in studying liquid-solid CE is achieving quantitative control over the charge generation, which requires an in-depth understanding of how these charge carriers are dictated by surface properties. Water has long served as a workhorse in extensive studies of liquid-solid CE due to its ready availability and inherent molecular polarity. It is worth mentioning that charge generation in water or at water-involved interfaces can arise not only from the widely known CE, but also from the other various manners, including introduction and conduction electrification 12 , 25 , as well as the charge transfer across the hydrogen bonds 26 – 28 . During liquid-solid CE, water is easily positively charged by solid surfaces 29 . Such a characteristic gives rise to the formerly prevalent ion-transfer model, in which anions (hydroxide ions) dissolved in water continually migrate to the solid surfaces due to their high surface affinity, especially those with hydrophobic or superhydrophobic properties, leaving excess positive charges (e.g., hydronium ions) in water 15 – 17 . Such ion-transfer model also incorporates the consideration of cations affinity toward the solid surfaces to explain the CE between acidic liquids and solid surfaces, denoting that ion propensity on surfaces is dependent on the pH values of liquids 30 , 31 . However, the ion-transfer model has been highly controversial due to debatable surface affinities of cations and anions in numerous experimental and simulation findings 28 , 32 – 36 , as well as its deficiency in explaining the recent findings that water can be either positively or negatively charged by a diverse array of solid surfaces (Fig. 1 ). Later, Wang et al. proposed that both electron and ion transfer are involved in liquid-solid CE, in which electron transfer dominates while ion transfer plays a subsidiary role 18 – 22 . Nevertheless, such a view fails to elucidate how electron transfer is dictated by surface properties, which is crucial for controlling both the polarity and magnitude of static charges generated in liquid-solid CE. Meanwhile, existing studies also overlook the possibility that transferred ions may come from liquid residues on surfaces 37 since exploited solid surfaces exhibiting hydrophilicity or low hydrophobicity (usually with a contact angle of <120 o ) inevitably adsorb liquids and ions contained 5 , limiting its applicability to the superhydrophobic surfaces that exhibit strong repellence to liquids. Fig. 1 Classification for the existing studies on liquid-solid CE based on the wettability of solid surfaces and charge polarity of water. There are three types of solid surfaces, including polymer (FEP, etc.) 18 , 22 , 51 , self-assembled monolayer (PFOTS-SiO 2 , etc.) 17 , 21 , 52 – 54 , and inorganic solid that is chemically active in water (SiO 2 , etc.) 20 , which are distinguished by three different font colors. The solid surfaces are located in the position of their corresponding wettability. The positive and negative areas represent the polarity of water charges generated during liquid-solid CE. The sign “?” means the area yet to be explored, i.e ., whether the water can acquire the negative charges from the superhydrophobic surfaces. Note that some works report mutually conflicting results regarding the charge polarity or the surface wettability. For example, SiO 2 is reported as hydrophobic in literature 21 (marked by a ), and water is positively charged by PP in literature 51 (marked by b ). In this work, we present novel insights into liquid-solid CE by designing superhydrophobic surfaces and studying underlying CE mechanisms. We design binary self-assembled monolayer of superhydrophobic surfaces by selecting a pair of mercaptan molecules with opposite electron-donating/accepting propensities, including 1H,1H,2H,2H-perfluorodecanethiol (FDT) and its fluorine-free analog, n-decanethiol (DT). By adjusting the molar ratio of FDT and DT, we are able to modulate the work function of the superhydrophobic surfaces due to the opposite dipole of FDT and DT 38 , 39 . Such superhydrophobic surfaces with regulable work functions enable unprecedented control of both the polarity and magnitude of static charges generated during CE. We reveal that the superhydrophobic surfaces can either positively or negatively electrify the liquids, filling the unexplored area in the field of liquid-solid CE (Fig. 1 ). We also find that the magnitude of static charges linearly varies with the work functions of the superhydrophobic surfaces, which indicates that the electron transfer between the contacted liquid and solid surface is driven by their work function differences. Such findings enable us to calculate the work function of water based on the corresponding values of the superhydrophobic surfaces. In addition, liquid-solid CE occurring on superhydrophobic surfaces eliminates the liquid residues on solid surfaces and accompanied ion transfer, standing in contrast to liquid-solid CE occurring on hydrophilic/hydrophobic surfaces, thus establishing a connection between surface wettability and ion transfer during CE.",
"discussion": "Discussion We design the binary self-assembled monolayer of superhydrophobic surfaces that possess regulable work functions and liquid repellence simultaneously. These two characteristics decouple the electrons and ions due to their respective functions in controlling electron transfer and excluding ion transfer during liquid-solid CE, leading to the intriguing phenomena of liquid-solid CE, as shown below. (1) There is a linear relationship between the work function of superhydrophobic surfaces and generated charges, which allows us to achieve, for the first time, quantitative control over the magnitude of static charges generated during liquid-solid CE. (2) The superhydrophobic surfaces can either positively or negatively electrify the water, salt, acid, and alkali aqueous solutions without deterioration in charging ability, denoting the independence of CE between liquids and superhydrophobic surfaces on the ion types and pH levels of liquids. In contrast, existing liquid-solid CE is subject to the pH values of liquids. A typical example is that the alkali solutions can only acquire negative charges from hydrophilic/hydrophobic surfaces during CE due to the continuous adsorption of OH − on the solid surface, and such ion adsorption will deteriorate the charging ability of the solid surfaces. In summary, we describe, quantify, and modulate the contact electrification between liquids and superhydrophobic surfaces, thereby revisiting the dynamics of liquid-solid CE. By integrating experimental findings and simulation results, we find that electron transfer, driven by the work function difference between the liquids and solid surfaces, underlies the liquid-solid CE. In addition, CE occurring on superhydrophobic surfaces excludes the ion transfer process, in contrast to the existing liquid-solid CE occurring on surfaces exhibiting hydrophilicity or low hydrophobicity, which proves that ion transfer during liquid-solid CE is affected by surface wettability. These findings advance the understanding of liquid-solid CE and open potential avenues for further exploration of practical applications. Furthermore, superhydrophobicity-induced unique results on liquid-solid CE are expected to promote the revaluation of previous findings in liquid-solid interfacial research, for example, interfacial chemistry under the situation of liquid flowing along a solid surface 49 ."
} | 2,400 |
38419632 | PMC10900513 | pmc | 6,078 | {
"abstract": "Species differentiation and the appearance of novel diversity on Earth is a major issue to understand the past and future of microbial evolution. Herein, we propose the analysis of a singular evolutive example, the case of microorganisms carrying out the process of anammox (anaerobic ammonium oxidation). Anammox represents a singular physiology active on Earth from ancient times and, at present, this group is still represented by a relatively limited number of species carrying out a specific metabolism within the Phylum Planctomycetota. The key enzyme on the anammox pathway is hydrazine dehydrogenase (HDH) which has been used as a model in this study. HDH and rRNA (16S subunit) phylogenies are in agreement suggesting a monophyletic origin. The diversity of this singular phylogenetic group is represented by a few enriched bacterial consortia awaiting to be cultured as monospecific taxa. The apparent evolution of the HDH genes in these anammox bacteria is highly related to the diversification of the anammox clades and their genomes as pointed by phylogenomics, their GC content and codon usage profile. This study represents a clear case where bacterial evolution presents a paralleled genome, gene and species diversification through time from a common ancestor; a scenario that most times is masked by a web-like phylogeny and the huge complexity within the prokaryotes. Besides, this contribution suggests that microbial evolution of the anammox bacteria has followed an ordered, vertical diversification through Earth history and will present a potentially similar speciation fate in the future.",
"introduction": "1 Introduction Anaerobic ammonium oxidation (anammox) ( Broda, 1977 ) is carried out by a specific group of bacteria with great significance in the global biogeochemical N-cycle ( Kuypers et al., 2018 ). Anammox bacteria accounts for a 30–70% of all N 2 released into the atmosphere ( Lam and Kuypers, 2011 ; Oshiki et al., 2016 ; Stein and Klotz, 2016 ). Thus, anammox bacteria have been widely used in wastewater treatment plants for the removal of fixed nitrogen loads ( Kartal et al., 2010 ; Stein and Klotz, 2016 ; Li et al., 2020 ) which indicates a major relevance in today’s world economy and sustainability. So far, the known anammox bacteria belong to the Phylum Planctomycetota ( Wang et al., 2019 ) which includes seven proposed candidate taxa based on 16S rRNA gene sequences: “Candidatus Brocadia,” “ Ca. Kuenenia,” “ Ca. Jettenia,” “ Ca. Scalindua,” “ Ca. Anammoximicrobium,” “ Ca. Anammoxoglobus” and “ Ca. Bathyanammoxibiaceae” ( Zhang and Okabe, 2020 ; Liao et al., 2022 ; Zhao et al., 2022 ). Because the culturing as monoespecific culture of these bacteria is arduous, today, most information on these bacteria has been obtained through whole-genome sequencing (WGS), including MAGs (Metagenome Assembled Genomes), of bacterial consortia and assemblages from nature or bioreactors ( Liao et al., 2022 ). The anammox process involves the use of nitrite or nitric oxide and ammonium to end by releasing N 2 to the atmosphere. The enzyme performing at the final step of the anammox pathway is hydrazine dehydrogenase (HDH) which represents the key enzyme of the process and it is present in all anammox-performing bacteria ( Maalcke et al., 2016 ; Akram et al., 2019 ; Liao et al., 2022 ). Hydrazine is a key intermediary ( Kartal et al., 2011 ) formed, today, mainly by a biotic process ( Dietl et al., 2015 ; Maalcke et al., 2016 ) or, in the ancient Earth, potentially by abiotic reactions ( Folsome et al., 1981 ; Jia et al., 2021 ). In spite of their relevance, scarce information is available on the evolutionary history of anammox bacteria. A recent study by Liao et al. (2022) suggests the origin of anammox bacteria on Earth around the Great Oxygenation Event, about 2.5 billion years ago. This places anammox bacteria close to the diversification of nitric reductase into the use of NO and O 2 as major substrates and the origin of aerobic respiration ( Ducluzeau et al., 2008 ; Santana et al., 2017 ). The transition from anaerobic respiration and the dominance of anaerobic processes (including denitrification) to an increasing significance of aerobic processes (including aerobic metabolisms) represented a major milestone in the history of the biogeochemical N-cycle on Earth. Specifically, as a consequence of increasing O 2 concentration, the diversification of ammonium oxidizing processes could appeared around that time, gaining importance the aerobic ammonium oxidizing microorganisms (including ammonium-oxidizing bacteria, ammonium-oxidizing archaea and commamox species carrying out the nitrification process) and likely limiting the expansion of the anammox bacteria restricted to anoxic niches including the oxygen-free side of anoxic-oxic boundaries. At their initials, anammox appeared to be related to anoxic, oxygen-poor, niches and environments ( Santana et al., 2017 ; Liao et al., 2022 ). Thus, the anammox bacteria constitute an ancient bacterial group maintaining their singularity through time ( Wang et al., 2019 ; Liao et al., 2022 ) and preserving their activity in spite of the numerous changes undergone on Earth. At present, anammox bacteria remain as a unified metabolic and phylogenetic bacterial group which are unique in their capability of carrying out the anammox process and directly releasing to the atmosphere around 30–70% of fixed nitrogen forms as N 2 ( Lam and Kuypers, 2011 ; Oshiki et al., 2016 ; Stein and Klotz, 2016 ). At present, microbial phylogeny and evolution present high complexity due to the huge microbial diversity existing on Earth ( Curtis et al., 2002 ). In addition, numerous processes are leading to horizontal gene transfer (HGT) in the prokaryotes, such as mobile genetic elements, viruses, transposition events, among others ( Johnston et al., 2014 ; Cuecas et al., 2017 ; Abe et al., 2020 ). Consequently, microbial phylogeny is often represented by web-like trees ( Doolittle, 2000 ; Hug et al., 2016 ) showing an increased level of complexity to account for the frequent HGT events occurring through evolution among the prokaryotes ( Goldman and Kaçar, 2022 ). Anammox can be selected as a singular group maintaining its uniqueness through evolution from ancient Earth. Thus, the anammox bacteria, and specifically their key HDH enzyme-encoding genes, can be an excellent case study for the analysis of gene, genome and species divergence through evolutionary history with perspectives to future diversification. The aim of this study is to analyze the divergence among the anammox bacteria as a unique metabolic and phylogenetic bacterial group that remains relatively independent of the rest of prokaryotes, as suggested by the related evolution of the HDH genes and the anammox bacterial genomes.",
"discussion": "5 Discussion The anammox bacteria represent a singular bacterial group showing relative isolation within the prokaryotes. So far, their evolutive history suggests that this group potentially evolves as a result of environmental constrains presenting minimum influences from other bacterial phyla such as through HGT-type for accelerated changes. As well, to our knowledge, no phages have been reported for the anammox bacteria perhaps a consequence of their low growth rate. These points suggest the existence of genomes showing less genomic plasticity than shown for most other bacterial phyla ( Johnston et al., 2014 ; Cuecas et al., 2017 ; Sheinman et al., 2021 ) although some genome variability within the anammox bacteria has been reported ( Ding and Adrian, 2020 ). While this is a hypothesis that remains to be tested, present information points to this putative scenario suggesting a study case for genomic analyzes and the evolutive history of this bacterial group. It is expected that future studies and additional microbial diversity surveys will greatly enhance the today’s relatively limited diversity within the anammox bacteria. Currently, a limiting sampling on anammox bacteria precludes to realize the actual diversity for the anammox bacteria existing on Earth ( Harhangi et al., 2012 ; Maalcke et al., 2016 ; Liao et al., 2022 ) in a similar way that the whole prokaryotic diversity on Earth remains to be truly understood ( Curtis et al., 2002 ). Potential issues with the slow growth rates of the anammox bacteria, potential discrimination during PCR amplification on microbial diversity surveys, added to the difficulty for culturing and the lack of monospecific cultures all sum up to make this singular bacterial group a difficult target for further research. Diversification and speciation among prokaryotes are major research lines to be developed in the years to come. It is expected that major advances on understanding these processes will be gained during the next decade or so. Herein, we propose that the anammox bacteria could represent a singular group, both on their metabolism and evolutive history, to analyze specific phenomena related to those issues within the prokaryotes. In the anammox bacteria, different aspects converge to point this group as a singular target for evolutive studies, most importantly, a time-located LCA, their slow evolution, a common genome-wide divergence trend and the apparently scarce influence on gene exchange from other bacterial groups. As pointed above, the environment, and specific niche requirements, might represent major evolutive factors influencing the divergence of the identified anammox clades. Currently, different niches has been reported for some of the different anammox clades which would confirm that the environment is having major influence on microbial evolution and specifically on the diversification of the anammox bacteria. Due to the high relevance of the anammox bacteria in the environment, including their application in waste treatment plants, great interest exist on understanding the physiology, ecology, evolution and behavior of this singular bacterial group. This perspective contributes to propose the anammox bacteria as a unique case study for the analysis of evolutive trends among bacteria and specifically within the anammox-performing bacteria. Current information, mostly from WGS and MAGs, suggests slow and independent evolution which appears to be mostly forced by environmental constrains. Future research on the development of these hypotheses will confirm the role and fate of anammox bacteria, and, from those results, the extracted knowledge con assist to comprehend the evolutive history of other bacterial phyla ( Goldman and Kaçar, 2022 )."
} | 2,652 |
39944239 | PMC11815889 | pmc | 6,079 | {
"abstract": "Abstract The rhizosphere is the soil region around plant roots hosting a diverse microbial community, influencing nutrient availability and how plants react to extreme conditions. However, our understanding of the fungi biodiversity and the impact of environmental variations on this biodiversity is still in its infancy. Our study investigates fungal communities’ diversity and functional traits in the rhizosphere of Nothofagus pumilio, one of the few winters deciduous treeline species in the world, forming the treeline in southern South America. At four distinct locations covering 10° latitude, we collected soil samples at treeline and 200 m below over four seasons during a single year. We employed ITS metabarcoding to elucidate fungal community structures. Our results reveal that fungal diversity was mainly determined by latitudinal variation, with higher levels during warmer seasons and lower altitudes. Interestingly, we found a marked dominance of ectomycorrhizal fungi at the treeline, particularly during the winter. In contrast, saprotrophic fungi were more abundant at lower altitudes, particularly during the warmer spring and summer seasons. These findings highlight the temporal and spatial dynamics of rhizospheric fungal communities and their potential roles in ecological processes, emphasizing the value of these communities as indicators of environmental change in high-elevation forests.",
"introduction": "Introduction The rhizosphere is the soil region surrounding plant roots hosting a diverse microbial community. This microbiome aids nutrient cycling, enhances plant growth through nutrient solubilization and hormone production, and influences plant defense mechanisms and plants’ response to extreme conditions [ 1 ]. Within this interface, fungi establish a variety of symbiotic relationships with trees, facilitating the uptake of nutrients through mycorrhizal associations [ 2 , 3 ], improving stress tolerance [ 2 , 4 , 5 ], and contributing to the overall stability and productivity of the forest ecosystem [ 6 ]. Among the key fungal guilds to this ecosystem are ectomycorrhizal (EcM) and saprotrophs, whose ecological roles are crucial in the forest [ 7–10 ]. EcM forms symbiotic associations with plant roots, which are fundamental to the tree’s nutrition and water stress response [ 11–13 ]. Saprotrophs significantly contribute to the decomposition of organic matter and nutrient cycling, which are essential for forest sustainability [ 14–16 ]. Nonetheless, our understanding of the factors influencing the composition of the rhizosphere and the diversity of associated organisms across different forest ecosystems, especially in the context of climate change, is still in its early stages of development [ 6 ]. In this context, seasonal variations and altitudinal gradients emerge as critical factors in examining the effects of climate change on rhizosphere composition and associated biotic communities [ 17–20 ]. The ‘treeline’ represents the ecotone where trees cease to grow at higher altitudes, marking the transition from forested to treeless elevations [ 21 ]. Notably, the treeline is influenced by the mean annual temperature and decreasing nitrogen levels with increasing altitude [ 22 ]. Around 70% of mountain treelines are currently experiencing shifts to higher elevations at an ~1.2 m per year rate, a value that increases to 3.1 m per year in tropical regions [ 23 ]. Thus, under the climate change scenario, treeline dynamics are intrinsically immersed in biodiversity conservation and mountain ecosystem management. Although the treeline’s extreme conditions and ecological importance have been recognised, the diversity and functionality of the rhizosphere fungal community in the treeline have not been systematically and comprehensively characterized [ 17 , 20 , 23 ]. The treeline of Southern South America Nothofagus Forests (SSAN forests, hereafter) is mostly dominated by Nothofagus pumilio, a deciduous tree species that can reach up to 30-m heights with an average lifespan of 200–300 years. N. pumilio can survive in low-temperature environments and thrive under reduced nutrient availability soil conditions [ 22 ]. This adaptation is reflected in its role as a dominant species in sub-Antarctic forests [ 24 , 25 ]. These interactions, particularly the EcM associations detected in the N. pumilio rhizosphere [ 26 , 27 ], indicate a symbiotic role between EcM and N. pumilio , facilitating survival and normal growth under unfavourable conditions [ 27 ]. In this sense, ~73%–79% of N. pumilio trees in the Argentinian Patagonia exhibit some EcM colonization [ 27 , 28 ]. Similar studies in Chilean Patagonia indicate that EcM- N. pumilio abundance increases with altitude [ 29 ]. However, this altitudinal pattern has only been reported in Tierra del Fuego during the summer season [ 29 ]. Consequently, it remains unclear whether these findings apply to other latitudes or seasons, such as the treeline, thereby limiting a comprehensive understanding of the role of EcM in the adaptation of N. pumilio to extremely cold environments. This limitation emphasizes the need to determine how complex fungal interactions contribute to the SSAN forest ecosystem resilience and health under climate change conditions. This study describes the dynamics of fungal communities in the N. pumilio rhizosphere at the treeline ecotone. We evaluated the effects of the altitude, latitude, and seasonal changes in the fungal biodiversity. We obtained rhizosphere samples from N. pumilio collected at four sampling points in Chilean Patagonia, considering two distinct altitudes (treeline and below-treeline) across the four yearly seasons. We examined the fungal community’s diversity through metabarcoding and provided evidence of the dynamic relationships between EcM and saprotrophic fungi across different environmental conditions. We hypothesize that EcM fungi dominate under extremely cold conditions at the treeline due to their potential ecological advantages under unfavourable conditions [ 27 ]. Consequently, we predict a higher relative abundance of EcM fungi at the treeline during winter compared to other seasons and altitudes, where saprotrophs are expected to be more prevalent. This research underscores the importance of monitoring fungal community dynamics as bioindicators for assessing the impacts of climate change on forest ecosystem resilience and function.",
"discussion": "Discussion Our study deepens the current understanding of the fungal ecology associated with the rhizosphere of N. pumilio at the treeline in Patagonian temperate forests. Employing a robust sampling design encompassing four latitudes, four seasons, and two altitudes, we observed a fungal community sensitive to each analyzed factor, revealing a dynamic spatiotemporal distribution of diversity and functions. Notably, our results suggest a relatively higher tolerance of EcM fungi to cold conditions than other guilds, as evidenced by their increased relative proportion during winter and treeline. More than 90% of forest trees in temperate and boreal zones form EcM associations [ 48 , 49 ], while most ground vegetation associates with arbuscular mycorrhizal or ericoid mycorrhizal fungi [ 48 , 50 ]. The structure of EcM communities varies with host species, temperature, geographic location, and soil properties, among other factors. However, the effects of season and altitude associated with temperature variations on the abundance and distribution of EcM have yet to be explored. Interestingly, we observed a higher relative abundance of EcM fungi under substantial snow cover in cold conditions [ 51 , 52 ]. Most research on extreme environments, such as the treeline, has not examined the influence of seasonal and altitudinal variation on the structure of the rhizospheric fungal community, thus providing only a partial picture [ 26 , 29 ]. The treeline represents a critical ecotone that is highly sensitive to climate change and environmental variations because it is controlled by low temperature [ 51 ]. Understanding the microbial dynamics at the treeline can provide insights into how ecosystem functions and biodiversity may respond to global climate change, offering essential information for conservation and management strategies [ 52–54 ]. Surprisingly, despite variations in alpha and beta diversity and taxonomic profiles among the four surveyed forests, the ecological roles represented by fungal guilds remained consistent between forests. This reflects the concept of functional redundancy, where diverse taxa can perform similar ecological functions, ensuring their persistence even under microbial biodiversity loss. In macroecological terms, this phenomenon has been explained as the maintenance of ecosystem functions by diverse species, reducing the likelihood of functional loss despite changes in community composition [ 55 , 56 ]. Examples of this phenomenon are seen in litter-decomposing fungal communities, where species maintain consistent nutrient cycling functions despite taxonomic shifts [ 57 , 58 ]. Similar redundancy has been observed in EcM fungal communities, where diverse taxa contribute equivalently to soil enzymatic activities, particularly in carbon and nutrient cycling, ensuring ecosystem functionality despite changes in fungal composition [ 59 ]. Furthermore, the consistent directional trends in ASV composition vectors across seasons ( Fig. 2C ) suggest that despite the geographic and altitudinal differences, fungal communities at each site undergo seasonal shifts similarly. This shared seasonal modulation implies that common environmental or biotic factors likely influence the variation in the rhizosphere fungal communities, indicating a degree of ecosystem stability in how fungal communities respond to seasonal changes, as similar patterns of variation are observed consistently across all sampling sites. Building on this, while site-specific differences in fungal diversity and composition were evident, soil conditions such as sulphur concentrations emerge as crucial environmental factors influencing fungal guilds ( Fig. S4 ). Our study highlights the interaction between sulphur concentrations and EcM abundance during the summer. This seasonal observation suggests that sulphur dynamics and availability, crucial for plant growth and ecosystem health [ 60 ], are strongly related to higher relative EcM abundance. Although these observations expand our understanding of EcM interactions with macronutrients in soil, the limitation to summer samples makes further seasonal studies necessary to fully capture the dynamics of these essential microbial processes throughout the year. The ubiquitous presence of Ascomycota and Basidiomycota across diverse geographies and environmental conditions underscores their ecological robustness, likely backed by a broad functional repertoire that enables their persistence in various environmental niches [ 61–63 ]. Notably, the seasonal and altitudinal patterns observed in this work, especially with Basidiomycota thriving at the treeline during winter, highlight their relative abundance under cold stress or perhaps their dominance due to the decrease in other fungal groups under such harsh conditions. This ability to endure extreme conditions may reflect a broader ecological strategy, where organisms capable of surviving in challenging environments take advantage of reduced competition in such niches [ 64 ]. Although tree metabolism is low during winter, a higher relative abundance of EcM may play a role in the nitrogen supply required by N. pumilio to endure the harsh conditions at the treeline in winter [ 65 ]. Remarkably, our analysis detected a significantly higher relative abundance of EcM at the treeline than just 200 m below treeline. This conspicuous change in EcM dominance occurring along such a narrow altitudinal range may respond to the fact that soil nitrogen availability steeply decreases with elevation and is significantly lower at the treeline than below it [ 66–68 ]. Indeed, previous studies demonstrated that a positive correlation between a higher relative abundance of EcM fungi and enzymatic capacity for organic N mobilization [ 65 ]. Thus, winter-active EcM, if confirmed, may benefit host trees during the harsh Patagonian winter and eventually explain the dominance of a winter deciduous species in the treeline, which is exceptional compared to most other treelines [ 22 , 69 ]. Other studies have shown that EcM not only survived in cold environments but was metabolically active and the most abundant guild in low temperatures conditions, even freezing [ 70 , 71 ]. A coherent pattern has also been described along an altitudinal gradient [ 72 ], Nevertheless, our study is the first one demonstrating the increasing relative abundance of EcM at the treeline, which is a worldwide limit of the tree growth form controlled by low temperatures. This result adds to the mounting evidence showing distinct altitudinal and seasonal patterns in fungal community composition under cold conditions. On the contrary, Koizumi et al. (2020) observed that summer temperatures in Pinus pumilia forests in Japanese archipelagos are the main parameter that forms the EcM community, particularly during the hottest summer months. However, they did not conduct winter samplings. Interestingly, they identified that EcM species such as Russula emetica , Suillus spraguei , Suillus sp. , and Tomentella sp . showed higher relative frequency as temperatures increases; while species like Cortinarius acutus , Cortinarius aurantiobasis , and Cortinarius junghuhnii showed higher relative frequency at lower temperatures [ 73 ]. Similarly, deep-snow treatments revealed that snow significantly reduced the number of EcM, although dominant genera like Cortinarius , Inocybe , and Tomentella were unaffected [ 74 ]. This revealed a correlation between low temperatures and EcM of the genus Cortinarius , which agrees with our findings. In our study, the Cortinariaceae family is the most abundant in winter and treeline, with the Cortinarius genus being the most abundant EcM in our samples. This finding agrees with a previous study that identified Cortinariaceae as the most abundant EcM in a plot at the treeline of N. pumilio across three years, using morpho-anatomic identification [ 26 ]. The Cortinariaceae family has been previously linked to trees of the Nothofagaceae family. Cortinariaceae was the most abundant family in N. pumilio seedlings in Argentinian Patagonia [ 75 ]. Similarly, in the southernmost tip of continental Chile, Cortinariaceae was among the most abundant in the rhizosphere of N. pumilio at different elevations [ 29 ]. In New Zealand, Cortinarius was the most abundant genus in the soil associated with N. menziesii [ 76 ]. This body of evidence suggests a key role for the Cortinariaceae family in the rhizosphere of N. pumilio in Patagonian temperate forests, especially under extremely low-temperature conditions. In summary, this study offers a compelling picture of spatiotemporal patterns of rhizospheric fungal communities in SSAN forests. Our findings demonstrate a significant prevalence of ectomycorrhizas during winter and in high-altitude regions at the treeline. The increased relative abundance of these symbiotic relationships during the harshest season and at this ecological edge suggests their potential importance in shaping rhizosphere dynamics under cold conditions. These results raise new hypotheses about the ecological role of EcM fungi in supporting plant life at the edge of Patagonian forests, which future studies can further explore."
} | 3,932 |
35340841 | PMC8942777 | pmc | 6,080 | {
"abstract": "It is anticipated that copper mining output will significantly increase over the next 20 years because of the more intensive use of copper in electricity-related technologies such as for transport and clean power generation, leading to a significant increase in the impacts on water resources if stricter regulations and as a result cleaner mining and processing technologies are not implemented. A key concern of discarded copper production process water is sulfate. In this study we aim to transform sulfate into sulfur in real mining process water. For that, we operate a sequential 2-step membrane biofilm reactor (MBfR) system. We coupled a hydrogenotrophic MBfR (H 2 -MBfR) for sulfate reduction to an oxidizing MBfR (O 2 -MBfR) for oxidation of sulfide to elemental sulfur. A key process improvement of the H 2 -MBfR was online pH control, which led to stable high-rate sulfate removal not limited by biomass accumulation and with H 2 supply that was on demand. The H 2 -MBfR easily adapted to increasing sulfate loads, but the O 2 -MBfR was difficult to adjust to the varying H 2 -MBfR outputs, requiring better coupling control. The H 2 -MBfR achieved high average volumetric sulfate reduction performances of 1.7–3.74 g S/m 3 -d at 92–97% efficiencies, comparable to current high-rate technologies, but without requiring gas recycling and recompression and by minimizing the H 2 off-gassing risk. On the other hand, the O 2 -MBfR reached average volumetric sulfur production rates of 0.7–2.66 g S/m 3 -d at efficiencies of 48–78%. The O 2 -MBfR needs further optimization by automatizing the gas feed, evaluating the controlled removal of excess biomass and S 0 particles accumulating in the biofilm, and achieving better coupling control between both reactors. Finally, an economic/sustainability evaluation shows that MBfR technology can benefit from the green production of H 2 and O 2 at operating costs which compare favorably with membrane filtration, without generating residual streams, and with the recovery of valuable elemental sulfur.",
"conclusion": "Conclusion A coupled hydrogenotrophic-aerobic MBfR system was optimized for sulfate removal and elemental sulfur production in mining-impacted water. By implementing automatic pH control the H 2 -MBfR achieved high average volumetric sulfate removals of 1.7–3.74 g S/m 3 -d at 92–97% efficiencies, close to performances reported for high-rate gas lift reactors but avoiding gas recycling and recompression and minimizing H 2 off-gassing risks. In addition, biomass accumulation was not a problem and H 2 supply was on-demand further simplifying gas management. The O 2 -MBfR achieved lower average S 0 formation performances of 0.7–2.66 g S/m 3 -d at 48–78% efficiencies instead, when compared to expanded bed or gas lift reactors. Both MBfRs can be further optimized, particularly the O 2 -MBfR, by automatizing the feed control and studying biomass and S 0 accumulation control measures. Finally, an economic evaluation shows that the coupled MBfR technology is cost-effective and that it can be also more sustainable based on current green hydrogen and oxygen price projections.",
"introduction": "Introduction It is expected that the current 21 Mt copper mining output will increase by 28% over the next 20 years, as the demand for copper from clean energy technologies grows by a factor of up to 2.7 in line with the Paris Agreement goals ( IEA, 2021 ). Copper intensity is significantly higher in electricity-related technologies for transport (24 kg/vehicle in conventional cars versus 53 kg/vehicle in electric cars) and power generation (1,100–1,150 kg/MW in natural gas-coal versus 2,800–8,000 kg/MW in solar photovoltaic-offshore wind), while expanded electricity networks will also need significant amounts of copper. Hence, the environmental impacts associated with copper mineral mining and processing are expected to rise accordingly unless environmental mining regulations become stricter and as a result, cleaner mining technologies are implemented. Particularly, the impacts of copper mining on water resources are twofold. The oxidation of residual metal sulfides in waste rock produces acid mine drainage characterized by acidic pH and high metals and sulfate concentrations ( Lottermoser, 2007 ; Dold, 2010 ). Similarly, sulfated process waters are commonly discarded along with mineral tailings in unlined surface impoundments ( Schwarz et al., 2020 ). Both effluents often contaminate surface and ground waters, and because of the large scale of some mining operations, the effects on wildlife and human health can be significant ( Simate and Ndlovu, 2014 ). To remove the sulfate present in mining effluents, biological treatment has been regarded as a more cost-effective option compared to chemical and physicochemical processes ( Skousen et al., 2019 ). Biological sulfate removal is a two-step process, involving an initial reduction of sulfate to sulfide, followed by the oxidation of sulfide to elemental sulfur (S 0 ). Because sulfide can be used as a ligand for sequential precipitation of metals and S 0 has agronomic value, biological treatment can be applied for resource recovery ( Kaksonen and Puhakka, 2007 ; Lin et al., 2018 ; Giordano et al., 2019 ; Kisser et al., 2020 ). With H 2 as the inorganic electron donor, the biological reduction reaction of sulfate to sulfide is ( Muyzer and Stams, 2008 ): \n 4 H 2 + S O 4 2 − + H + → H S − + 4 H 2 O Δ G 0 ′ = − 151 . 90 kJ / reaction ( − 19 .0 kJ / e − ) \n (1) \n \n H 2 is a good electron donor substrate for the treatment of inorganic sulfate-rich waters because it is cheap ( Bijmans et al., 2011 ), clean and non-toxic, and can be produced on-site using green electricity ( Acar and Dincer, 2018 ). Furthermore, H 2 utilization efficiency can be optimized for sulfate reduction by limiting the CO 2 feed to control methanogenesis ( van Houten et al., 2009 ). With O 2 as the electron acceptor, the sulfide-oxidation reactions can be summed up as follows ( Madigan et al., 2019 ): \n H S − + H + + 0.5 O 2 → S 0 + H 2 O Δ G 0 ′ = − 209 . 4 kJ / reaction ( − 104 . 7 kJ / e − ) \n (2) \n \n \n S 0 + 1.5 O 2 + H 2 O → S O 4 2 − + 2 H + Δ G 0 ′ = − 587 . 1 kJ / reaction ( − 97 . 9 kJ / e − ) \n (3) \n \n \n H S − + 2 O 2 → S O 4 2 − + H + Δ G 0 ′ = − 796 . 5 kJ / reaction ( − 99.6 kJ / e − ) \n (4) \n \n Reaction 2 of incomplete oxidation of sulfide to S 0 is the desirable reaction as sulfide is not reoxidized to sulfate and only 25% of the O 2 is consumed, and because S 0 is a valuable end product. S 0 formation is favored under O 2 limitation (∼0.1 mg O 2 /L), and S 0 yields >90% can be reached ( Lin et al., 2018 ). Dissimilatory sulfate reduction ( Eq. 1 ) is mostly catalyzed by sulfate-reducing bacteria (SRB) belonging to the phylum Desulfobacterota ( Waite et al., 2020 ). Particularly the genus Desulfovibrio is often dominant in sulfate-reducing reactors ( Dar et al., 2007 ; Schwarz et al., 2020 ) under pH neutral conditions, though in acidic environments, species within the Peptococcaceae family of the Firmicutes phylum have been described ( Sánchez-Andrea et al., 2015 ). Sulfide oxidation ( Eqs. 2 - 4 ) used in biotechnological applications, on the other hand, commonly involves sulfur-oxidizing bacteria (SOB) of the genera Thioalkalimicrobium , Thioalkalivibrio , or Thiobacillus ( Janssen et al., 1995 ; van den Bosch et al., 2007 ; Sorokin et al., 2013 ; Muyzer et al., 2013 ). While sulfate removal systems have been well studied and even commercial systems exist ( Kaksonen and Puhakka, 2007 ; Hao et al., 2014 ), they continue to be optimized ( Mora et al., 2020 ). New applications are also constantly emerging including the fluidized bed membrane bioreactor ( Oztemur et al., 2020 ), the membrane biofilm reactor ( Schwarz et al., 2020 ), and the sulfur-packed bed reactors with excess sulfate rejection by nanofiltration ( Asik et al., 2021 ). Particularly, the membrane biofilm reactor (MBfR) ( Nerenberg, 2016 ; Rittmann, 2018 ) is promising because it makes efficient delivery of gaseous substrates possible (H 2 and O 2 in Eqs. 1 and 4 ). The MBfR is made up of bundles of hollow-fiber membranes that are hydrophobic and non-porous. A gaseous substrate is supplied to the lumen of the fibers and transferred by diffusion into a biofilm growing on the outer surface of the membrane ( Zhou et al., 2019 ). Table 1 summarizes reported sulfur transformation performances involving gaseous substrates of selected studies focused on sulfur removal. Mining operations require high-rate microbial processes because of the large process-water and effluent flows involved. MBfRs, fluidized bed reactors (FBRs), and gas lift reactors (GLRs) are more commonly used with gaseous substrates ( Di Capua et al., 2015 ; Sinharoy et al., 2020 ). Because FBRs and GLRs deliver substrate gases by bubbling, they require gas recompression and recycling and have a higher risk of H 2 and H 2 S off-gassing. The high cost of membranes has been regarded as the main obstacle for MBfR adoption instead ( Martin and Nerenberg, 2012 ), however, the cost of commercial membranes is decreasing and new commercial applications will continue to emerge ( Nerenberg, 2016 ). TABLE 1 Examples of sulfate-reducing and sulfide-oxidizing systems focused on sulfur removal. Wastewater Substrate Reactor type, liquid volume, operating temperature Average sulfide or sulfur productivities (kg S/m 3 -d) and efficiencies (in parenthesis) Reference \n Sulfate-reducing reactors \n Synthetic 80% H 2 /20% CO 2 \n GLR/Pumice carrier, 4.5 L, pH 7–7.5, 30°C 4.67–7.07 (77–82%) \n van Houten et al. (1994) \n \n van Houten et al. (1995) \n \n van Houten et al. (1996) \n Cu tailings water 80% H 2 /20% CO 2 \n MBfR, 25 ml, pH 7.6, 21°C 0.81 (98%) \n Schwarz et al. (2020) \n Cu tailings water 95% H 2 /5% CO 2 \n MBfR, 25 ml, pH 8.0 ± 0.2, 21 ± 3°C 1.70–3.74 (97–92%) This study \n Sulfide-oxidizing reactors \n Synthetic Air Expanded bed, 12 L, pH 7.2–7.6, room temp 5.00 (72%) \n Janssen et al. (1997) \n Sulfidogenic reactor effluent O 2 \n MBfR, 43 ml, pH 7–9, room temp 2.40 (76%) \n Sahinkaya et al. (2011) \n Synthetic Air GLR, 4.9 L, pH 7.6–8, room temp 2.91 (79%) \n Lohwacharin and Annachhatre, (2010) \n Synthetic Air MBfR, 4.1 L, pH 7.5, 30°C 2.0–5.5 (83.7–56.3%) \n Jiang et al. (2019) \n H2-MBfR effluent Air/O 2 \n MBfR, 25 ml, variable pH and pH 8.0 ± 0.2, 21 ± 3°C 0.70–2.66 (78–48%) This study A critical operational aspect of the coupled reducing and oxidizing processes is pH control. A pH that is too low increases the risk of H 2 S toxicity and one that is too high increases the risk of CaCO 3 scaling ( Suárez et al., 2020 ). To obtain the overall net acid-base demand of the coupled process, the acid-base equivalents in the key reactions must be summed up. Reaction 1 consumes one equivalent of strong acid (H + ) per mole of sulfate reduced, while reaction 2 also consumes one equivalent of strong acid per mole of S 0 formed. Hence, under optimal conditions of 100% S 0 formation, the acid demands of the reducing and oxidizing reactors approach the 1:1 ratio. This is an approximation because in the normal working alkaline pH range some H 2 S in Eqs 1 , 2 is unionized and this fraction varies with pH. In addition, reaction 3 shows that further oxidation of S 0 to SO 4 \n 2- , instead of consuming acid, produces 2 equivalents of strong acid per mole of S 0 oxidized. These insights are critical to the correct design of an automatic pH control strategy, indicating the requirement for acid dosing in the reducing module, and acid and eventually base dosing in the oxidizing module. Also, the preceding acid-base accounting is valid for pH \n ∼ \n 8, the chosen operational pH, in which HS − is dominant and H 2 S toxicity is minimized. Other factors and MBfR processes can also affect the pH, such as feed water alkalinity, external CO 2 gas addition, CaCO 3 precipitation/dissolution, CO 2 consumption by autotrophs, and CO 2 production by heterotrophs ( Tang et al., 2011 ). However, it is expected that these processes will be insignificant given the high metabolic rate for sulfur species and considering that the CO 2 ratio in the gas mixture is kept low and that the pH of \n ∼ \n 8 minimizes the risk of CaCO 3 scaling. Nevertheless, a pH model should be developed to assess the relevance of these processes under different operating conditions ( Xia et al., 2016 ). Different pH control strategies have been implemented in MBfRs such as acid injection without automatic control ( Schwarz et al., 2020 ), and CO 2 dosing as part of the reactive gas mixture ( Suárez et al., 2020 ) or with a separate membrane ( Xia et al., 2020 ). It has been shown that acid/base injection lacking automatic control is prone to overshooting and that CO 2 dosing has limitations for influents rich in Ca such as mining effluents since the risk of CaCO 3 scaling increases. Hence, the chosen alternative in this study is acid injection with automatic control for both reducing and oxidizing modules. Although the oxidizing module might demand a base when it is underperforming, this situation can be theoretically avoided by careful O 2 dosing. The main purpose of this research was to optimize the operation of coupled reducing and oxidizing MBfR modules for conversion of sulfate-sulfur into elemental sulfur from sulfate-rich mining process water. We assessed the effect of increasing sulfate surface loading, automatic pH control, and hydrogen and oxygen gas pressures, on the removal of sulfate, the amount of S 0 produced, and pH stability.",
"discussion": "Results and Discussion Performance of the H 2 -MBfR As can be seen from the operational conditions of Table 2 ; Figure 2A , the experimental strategy consisted of gradually increasing the sulfate loading from 4.0 to 10.5 g S/m 2 -d in 4 phases. During each phase optimizations of gaseous pressures were carried out, first adjusting the pressure of the H 2 /CO 2 mixture for the efficient reduction of sulfate, and then that of air and later O 2 for the effective transformation of S 2- to S 0 . Figure 2A further shows the variation of pressures during the experiment and Table 3 the performances and obtained fluxes. FIGURE 2 \n (A) Operating conditions and evolution of SO 4 \n 2− and S 2− from the H 2 -MBfR and O 2 \n − -MBfR; (B) evolution of SO 4 \n 2− and S 2− from the reducing stage (H 2 -MBfR) and (C) evolution of SO 4 \n 2− and S 2- from the oxidizing stage (O 2 \n − -MBfR). TABLE 3 Performances and fluxes. Parameter/Operating phase F1 F2 F3 F4 \n Average performances (%) \n H 2 -MBfR SO 4 \n 2- reduction \n a \n \n 62 97 95 92 H 2 -MBfR S 2- formation \n a \n \n 57 95 95 88 O 2 -MBfR S 2- oxidation \n b \n \n 88 96 88 78 O 2 -MBfR S 0 production \n c \n \n 89 69 65 62 Overall SO 4 \n 2- removal \n a \n \n 53 68 66 65 Overall S 0 production \n a \n \n 47 65 55 46 \n Average fluxes (g/m 2 -d) \n SO 4 \n 2- -S 2.33/−0.11 \n d \n \n 4.32/−0.64 \n d \n \n 5.74/−0.79 \n d \n \n 9.54/−1.19 \n d \n \n S 2- -S −2.27/1.02 \n d \n \n −4.23/1.92 \n d \n \n −5.73/2.44 \n d \n \n −9.19/3.84 \n d \n \n S 0 -S \n e \n \n −0.91 −1.28 −1.67 −2.66 H 2 \n 0.63 1.07 1.44 2.47 O 2 \n 0.74 2.24 2.81 4.30 a % of influent sulfur. b % of sulfide formed. c % of sulfide oxidized. d Fluxes in H 2 -MBfR and O 2 -MBfR, respectively. e Specific surface area of the O 2 -MBfR is 96% higher than that of the H 2 -MBfR which explains the relatively low S 0 -S fluxes. During phase 1, the sulfate load was 4 g S/m 2 -d. At the beginning of the phase, the reactor experienced H 2 limitation at an H 2 /CO 2 pressure in the 2-3 psig range. Consequently, only 23% of the sulfate was removed. To increase the sulfate removal rate, starting on day 3, the H 2 /CO 2 pressure was increased three times up to 10 psig during the first 21 days. The reactor responded to this stepped increase in pressure with the total reduction of sulfate on day 24. Considering that the increase in pressure could have been excessive, at the end of stage 1 and the beginning of stage 2, the H 2 /CO 2 pressure was gradually decreased to 7 psig, and indeed, no increase in the effluent sulfate concentration was noticed. During phase 1, an average sulfate reduction efficiency of only 62% was obtained, because of the extended H 2 limiting condition. However, due to the rapid response of the reactor to the increase in pressure of H 2 /CO 2 , before the end of phase 1 (day 24), 100% sulfate removal at a flux of 4.07 g S/m 2 -d was achieved. In phase 2, the reactor quickly adapted to a 12.5% increase in sulfate loading to 4.5 g S/m 2 -d, not being necessary to increase the H 2 /CO 2 pressure of 7 psig. Only a slight temporary increase in sulfate concentration was recorded at the beginning of the phase. Then, a steady-state was reached characterized by a stable production of sulfide, reaching a high average removal of sulfate of 97% (4.32 g S/m 2 -d) in the phase. Furthermore, according to Figure 2B , if the initial sulfate peak was eliminated, sulfate removal would have averaged 100%. This stability and efficiency of the H 2 -MBfR can be considered a significant strength of the reactor system. During phase 3 the sulfate SLR was further increased by 33.3% to 6.0 g S/m 2 -d while keeping the H 2 /CO 2 pressure constant at 7 psig. The two specific increases in the effluent sulfate concentration at the beginning of this stage, which are again due to the adaptation of the biofilm to the new load, explain the loss of sulfate removal efficiency, which averaged only 95% during phase 3. However, the average sulfate flux increased to 5.74 g S/m 2 -d during this phase. It should be noted that although the combined load increase during phases 2 and 3 was 50%, a permanent increase in H 2 /CO 2 pressure was not necessary to satisfy the increased demand for H 2 . This shows that in the H 2 -MBfR the delivery of gas by the membrane is demand-driven, which means that gases within certain reasonable pressure ranges are never overdosed ( Rittmann, 2018 ). Finally, in phase 4 the SLR was increased by 75% to 10.5 g S/m 2 -d, by increasing the inflow, calculated to achieve an HRT of 3 h. Again, after an initial peak of adaptation, the effluent sulfate stabilized at values close to zero. To control the initial peak of effluent sulfate in the face of the significant increase in SLR this time, the H 2 /CO 2 pressure was temporarily increased to 10 and then to 12 psig. Possibly, the control of the sulfate peak would have been more effective if the H 2 /CO 2 pressure increase had coincided with the load increase. Once again, the good performance of the H 2 -MBfR was reflected during this stage in the high average sulfate reduction efficiency of 92%, only affected by the initial sulfate peak. During the final phase of the experiment, an average sulfate flux of 9.54 g S/m 2 -d was reached, which represents a very important advance to values of previous MBfR investigations of <2.74 g S/m 2 -d ( Ontiveros-Valencia et al., 2012 , 2016 ; Schwarz et al., 2020 ; Suárez et al., 2020 ). Likewise, the average H 2 consumption rate of 2.47 g H 2 /m 2 -d obtained during phase 4 also exceeds the values of the MBfR literature of <0.68 g H 2 /m 2 -d ( Zhao et al., 2013a ; Zhao et al., 2013b ; Schwarz et al., 2020 ), considering a variety of electron acceptors. This demonstrates the value of using membranes with higher gas permeability in our study. \n Figures 3A,B shows the influent and effluent pH and ORP variations of the MBfRs. In general, good performance of the pH control system is observed in the H 2 -MBfR. In turn, the ORP of the H 2 -MBfR remained practically throughout the experiment below -200 mV, in the appropriate range for SRBs, and most of the time, even in the optimal range of < −270 mV ( Hao et al., 2014 ). FIGURE 3 Evolution of (A) pH and (B) ORP from the H 2 -MBfR and O 2 -MBfR. As Table 1 shows, GLRs have achieved the highest volumetric sulfate reduction rates with H 2 , however at steady-state efficiencies of only around 80% in the 7–7.5 pH range, which would not allow complying with the strictest sulfate water-quality standard of 250 mg/L ( INN, 1978 ; European Union, 1998 ; US EPA, 1999 ). Nevertheless, van Houten et al. (1995) reached a close to 100% efficiency at pH 8 for a short period, before the reactor failed due to clogging. In this study, the MBfR also achieved high sulfate reduction rates and efficiencies and further performance increases should be tested based on GLR data. Most research on hydrogenotrophic microbial reduction of oxyanions such as sulfate has focused on denitrification ( Karanasios et al., 2010 ; Di Capua et al., 2015 ). As this study showed, biomass accumulation problems reported in high-rate denitrification MBfRs ( Di Capua et al., 2015 ) will be minimal in the sulfidogenic H 2 -MBfR because with H 2 the biomass yield of sulfate reduction is about one-third of that of denitrification ( Rittmann and McCarty, 2001 ). Performance of the O 2 -MBfR The operation of a sulfide oxidizing bioreactor is complex because only one control variable, the air or oxygen supply flux, is available to achieve two competing goals, maximizing sulfide oxidation and the formation of S 0 . On the one hand, increasing the oxidation of sulfur requires increasing the supply of oxygen, and on the other, it is necessary to limit the supply of oxygen to favor the formation of S 0 . The efficiency of sulfide oxidation was evaluated by measuring the sulfide concentration and that of S 0 formation by measuring the sulfate concentration. Sulfide in the effluent was considered as a sign of insufficient oxygen and the formation of sulfate as a sign of excess oxygen. With this criterion, an attempt was made to optimize the operation of the O 2 -MBfR, a task that was complicated by the programmed changes in the H 2 -MBfR operation. \n Figure 2C shows the influent and effluent sulfide and sulfate concentrations of the O 2 -MBfR during the experiment. During the first half of phase 1, the effluent sulfide was near zero and the influent and effluent sulfate concentrations were similar, indicating that the 3 psig air pressure was optimal. Then, coinciding with the start of the increase in influent sulfide on day 14, sulfide began to be detected in the effluent, so that during the second half of phase 1 the air pressure was gradually increased from 3 to 12 psig. During phase 1, the reactor performed well, with oxidation efficiencies of S 2- of 88% and of production of S 0 of 89%, which during the first 2 weeks were even higher (97 and 89%, respectively), while the S 2- and S 0 fluxes averaged 1.02 and -0.91 g S/m 2 -d, reaching maximums of 1.54 and -1.47 g S/m 2 -d on day 24. The O 2 -MBfR performed very well again during the first 2 weeks of phase 2 with S 2- oxidation efficiencies of 95% and S 0 production efficiencies of 91%. As the manufacturer’s recommended maximum operating pressure of 10 psig began to be exceeded, on day 31 the air supply was changed to pure oxygen, generating an increase in oxygen pressure from 2.7 psig (in the air) to four psig. The increase in the effluent sulfate concentration beginning on day 40, indicates an excess of oxygen so that the oxygen pressure was gradually decreased to 2.5 psig between days 46 and 52, achieving the desired sulfate decrease from 343 to 43 mg S/L on day 56 but with an increase in sulfide from 7 to 69 mg S/L. The sulfate and sulfide concentrations decreased to 0 and 3 mg S/L, respectively, on day 59 due to a decrease in influent sulfide. Therefore, small variations in the concentration of the influent sulfide can alter the redox balance, improving or deteriorating the performance of the reactor, requiring continuous adaptation of the oxygen pressures to the variations in the sulfide load. In phase 2, due to the excessive supply of oxygen, the average efficiency of sulfide oxidation improved slightly compared to the previous stage (96%) while the average efficiency of S 0 production significantly worsened (69%). Due to the increase in the sulfide load, however, the average S 2- and S 0 fluxes increased to 1.92 and −1.28 g S/m 2 -d, respectively. Phase 3 was characterized by a significant increase in the sulfide load, for which an increase in oxygen pressure from 2.5 to 3.5 psig was carried out at the beginning of the phase (day 60). Initially, as the H 2 -MBfR was slow to respond to the increase in sulfate loading with a higher production of sulfide, the increase in oxygen pressure generated an over-oxygenation condition in the O 2 -MBfR. As a result, the effluent sulfate increased significantly, which could be controlled by reducing the O 2 pressure to 1.5 psig between days 78 and 80. At this oxygen pressure, the oxidizing capacity was insufficient, generating a rapid increase in the effluent sulfide, and therefore the pressure was gradually increased to four psig from day 87 to day 98 when a minimum of sulfate and sulfide effluent of 7 and 13 mg S/L was achieved. However, towards the end of phase 3, there was a slight increase in the effluent sulfate that was not eliminated before the start of phase 4. Because with each adjustment of the oxygen pressure carried out, either too oxidizing or too reducing conditions were generated during phase 3, without achieving a stable optimum point, the oxidation efficiencies of S 2- and formation of S 0 reached values of only 88 and 65%, respectively, although the corresponding average fluxes continued to increase (2.44 and 1.65 g S/m 2 -d). Finally, in phase 4, faced with the increase in the influent load of sulfate to the H 2 -MBfR, an operating strategy different from that of the previous phase was tried, leaving the O 2 pressure constant during the first 2 weeks, pending that the H 2 -MBfR responded to the increase in sulfate loading with an equivalent increase in sulfide flux. Only on day 118, when a high level of sulfide was detected in the O 2 -MBfR effluent, the O 2 pressure began to be increased from 3 psig to five psig on day 127, but without achieving a decrease in the effluent S 2- . Therefore, the O 2 pressure was successively increased to 8 psig on day 133, and then to 10 psig on day 137, until the sulfide disappeared on day 139. During this phase, average efficiencies of oxidation of S 2- of only 78% and production of S 0 of 62% were achieved, although they were obtained at maximum average fluxes of S 2- of 3.84 g S/m 2 -d and S 0 of −2.66 g S/m 2 -d. There is the potential to increase these fluxes significantly, as Sahinkaya et al. (2011) reported S 0 fluxes of −9.6 to −48 g S/m 2 -d at O 2 pressures of 3–15 psig using MHF 200 TL membranes (Mitsubishi Rayon). During the first 3 phases, the O 2 -MBfR did not have a pH control system, so it varied freely. The production of S 0 consumes 1 mol of weak acid for each mole of S 0 produced, so the pH tends to rise when the efficiency of S 0 production is high ( Eq. 2 ) and to decrease when sulfate production dominates ( Eq. 3 ), as shown in Figure 3A . It was considered that S 2- loading increases could cause pH increases and generate fouling of the membrane due to precipitation of CaCO 3 on the membrane, so on day 98, an automatic pH control system was installed. In Figure 3A it can be seen that the pH of the O 2 -MBfR became more stable after installation. According to Lin et al. (2018) , ORP levels from −400 to −137 mV are optimal for the formation of S 0 . Indeed, when ORP levels exceeded the −137 mV limit twice, on days 45–49 (34 mV > ORP > -46mV) and days 77–80 (−86 mV > ORP > −89 mV), the worst yields of S 0 formation were obtained. These two events were further characterized by high sulfate concentrations and low pH levels. However, these two very time-limited events do not explain the observed levels of S 0 formation inefficiency, so it remains to be determined, which are the optimal levels of ORP in O 2 -MBfR reactors for S 0 formation. Overall Performance of the MBfR System The best average global transformation yield of influent sulfate into elemental sulfur was 65%, achieved during phase 2. This phase was characterized by a sulfate reduction process that was stable and also had the highest average yield (97%). This stability of the H 2 -MBfR could also contribute to the oxidation efficiency of the O 2 -MBfR being the highest (96%). As mentioned before, the exchange of air for oxygen affected the efficiency of S 0 formation (69%), explaining the overall performance of phase 2. Specifically, based on data from days 34 and 38 of phase 2, the average global transformation yield reached 98%. During phase 3, on days 91–105, a good overall performance of 85% transformation from SO 4 \n 2- -S to S 0 was achieved. Also, during stage 4, on days 134–147, the overall transformation yield from SO 4 \n 2- -S to S 0 was 74%. Based on these good punctual results, future studies should evaluate automatic oxygen pressure control strategies in the O 2 -MBfR, based on the ORP for example, because the process of S 0 formation is very sensitive to this variable. Also, the recovery of S 0 particles from the O 2 -MBfR must be still systematically evaluated. As the MBfR modeling study of Jiang et al. (2019) showed, S 0 can make up to 80% of the biofilm dry weight if it is assumed that S 0 remains attached to the cells once excreted. Bacterial Community Diversity The majority of the sequences in the anaerobic module were affiliated mainly to two genera, Desulfomicrobium and Desulfovibrio , both from the order Desulfovibrionales ( Kuever, 2014 ), which members are known as sulfate-reducers. Their relative abundance was 77.6 ± 1.6% at the end of the operation, where sulfate removal was completely achieved ( Figure 4 ). The effluent collected in the H 2 -based MBfR showed that the dominant phylotypes were from the same SRB genera, although their abundance decreased to 63.2% while those of the Sulfurospirillum genus increased the most from 1.6 ± 0.2–8.8%. This genus consists of versatile, often microaerophilic bacteria, growing with many different substrates where electron donors can be hydrogen, sulfide, and organic acids, while electron acceptors under anaerobic respiration are nitrate, fumarate, and sulfur species other than sulfate (e.g., thiosulfate, elemental sulfur, polysulfides) ( Goris and Diekert, 2016 ). Dissolved oxygen in the influent or diffused through the flexible tubing could perhaps result in sulfur partial oxidation products which were then reduced to sulfide by Sulfurospirillum ( Lin et al., 2018 ). This overrepresentation of the Sulfurospirillum genus in the effluent compared to the fibers could then be due to flexible tubing biofilms. Carbon dioxide utilization is not a physiological feature found in Sulfurospirillum spp., though its growth can be promoted by the soluble microbial products (SMP) released by the SRB ( Ontiveros-Valencia et al., 2018 ; Schwarz et al., 2020 ). Besides, Sánchez-Andrea et al. (2020) demonstrated recently that Desulfovibrio ssp. secrete formate and acetate while growing autotrophically with H 2 and sulfate. Hence, Sulfurospirillum could have also grown anoxically with both formate and H 2 as electron donors; acetate as the carbon source; and influent nitrate (0.13 mM) in the H 2 -MBfR, and S 0 in the O 2 -MBfR, as electron acceptors ( Stolz et al., 2015 ). Finally, in a high-rate S 0 reducing system to treat sulfate-rich metal-laden wastewater ( Sun et al., 2018 ), the predominant sulfidogenic bacterium was also Desulfomicrobium . With just S 0 , Sufurospirillum was initially dominant but as high-rate sulfate feeding (at levels comparable to our study) began, Desulfomicrobium replaced Sulfurospirillum . FIGURE 4 Relative abundances at the genus level for the reducing stage of the sequential system (H 2 -MBfR), obtained from an effluent sample and three sections of fiber and for the oxidizing stage (O 2 -MBfR), obtained from an effluent sample and three sections of fiber taken at the end of the operation. Fiber section order in both cases is 1, 2, and 3 from bottom to top, Introduction being closest to the gas feed. In the sulfide oxidation stage, the bacterial population composition showed significant changes, particularly in Introduction , Materials and Methods exposed to the higher pressure of O 2 , where the most represented phylotypes were related to Hydrogenophaga , with a relative abundance of 38 and 46% respectively (42.1 ± 5.4% overall). Described members of this genus are chemo-organotrophic or chemolithoautotrophic, growing by the oxidation of H 2 with CO 2 as a carbon source ( Willems and Gillis, 2015 ); therefore, its growth in the sulfide oxidation stage may be due to carrying over of H 2 /CO 2 to the oxidizing biofilm. However, this genus may also have a key role in oxidizing reduced sulfur species. Hydrogenophaga spp. have been identified as members of H 2 S oxidizing communities in wastewater treatment ( Cytryn et al., 2005 ; Vannini et al., 2008 ; Li et al., 2020 ) and described as a dominant SOB in a full-scale H 2 S-bioscrubber (7.45% relative abundance) ( Haosagul et al., 2020 ). Recently, colorless SOBs were characterized in environmental samples using functional marker genes ( Luo et al., 2018 ; Jaffer et al., 2019 ). Several sulfide oxidation pathways for conserving energy and S 0 formation have been described, including the sulfur oxidation (Sox) and dissimilatory sulfite reductase (Dsr) systems ( Madigan et al., 2019 ), and sulfide quinone oxidoreductase (SQR) and flavocytochrome c (Fcc) systems ( Dahl, 2020 ). These systems or their elements are universally distributed among sulfur chemolithotrophs due to lateral gene transfer and pathways can even be redundant ( Dahl, 2020 ). Jaffer et al. (2019) showed that most of the soxB gene clone sequences were affiliated to the genus Hydrogenophaga , while Luo et al. (2018) found that soxB and sqr genes were also predominantly expressed in Hydrogenophaga . On the other hand, the genus Brevundimonas , capable to grow by using organic carbon released by autotrophic bacteria and determined previously in an anoxic biotrickling filter for H 2 S removal ( Khanongnuch et al., 2019 ) and an airlift bioreactor for biogas desulfurization (4.38% relative abundance) ( Quijano et al., 2018 ) was second in abundance in the sulfide oxidation stage (10.3 ± 0.2% considering Introduction , Materials and Methods ). Interestingly, in Results and Discussion and more pronounced in the effluent of the oxidizing module, SRB phylotypes belonging to the genera Desulfomicrobium and Desulfovibrio carried over from the reducing stage were dominant (28.4 and 41.0% abundance, respectively) which may be closely associated with the depletion of oxygen, promoting reducing conditions. On the contrary, Hydrogenophaga phylotypes reached only 7.7% in Results and Discussion of the oxidizing module and 5.4% in the effluent. Treatment Economics and Sustainability The electron donor cost makes up about one-half of the operational cost of sulfate reduction, giving costs of 0.26 and 0.20 US$/kg SO 4 for ethanol and H 2 , respectively ( Bijmans et al., 2011 ). Besides being cheaper, as solar or wind-based production systems become widespread, green H 2 will be also more sustainable ( Acar and Dincer, 2018 ). Green H 2 is one of the key fuels that will help tackle the critical energy challenges of a wide range of sectors including long-haul transport, chemicals, iron and steel, and storage of electricity from renewables ( IEA, 2019 ). Consequently, countries worldwide are investing heavily in the research and development of green hydrogen-based technologies for emissions reduction ( UNEP, 2020 ). Armijo and Philibert (2020) estimate a very competitive short-term cost of green H 2 for Chile of around 2 US$/kg H 2 (both solar and wind-based), in the range of 1.60–2.05 US$/kg H 2 reported for coal-based production with carbon capture and storage in Canada ( Yu et al., 2021 ). Considering that 4 mol of H 2 are required to reduce each mole of sulfate, a green H 2 cost of 0.17 US$/kg SO 4 is obtained (equal to 0.27 US$/m 3 if the sulfate content of the tailings water of 1.5 g/L is considered). Since H 2 is the main operating cost item of reducing MBfRs, this value compares favorably with the cost for technologies such as reverse osmosis of 0.5–2.5 US$/m 3 ( World Bank, 2019 ) which in addition generates a residual stream. It must also be considered that for the medium and long term a significant reduction in the cost of H 2 is expected because of technological development and its mass production ( IRENA, 2020 ). The production cost of green hydrogen depends mainly on the cost of the renewable power, the intermittency of its supply, and the cost of the electrolyser ( CEFC, 2021 ). Furthermore, transport and storage requirements may have to be factored in the cost of delivered hydrogen. The electrolyser CAPEX is also dependent on the installed capacity, varying from 450 to 850 US$/kW for input energies of 100 and 2 MW in the case of alkaline electrolysers, respectively ( Proost, 2019 ). We estimate that a 100 L/s desulfurization plant for a large tailing facility would demand an energy input for onsite H 2 production of 2 MW, having then an electrolyser CAPEX of 850 US$/kW, while the above reported cost of green H 2 for Chile assumes an electrolyser CAPEX of 600 US$/kW. For smaller electrolysers the CAPEX in US$/kW varies even more with scale ( Proost, 2019 ). The cost of HCl is not negligible but amounts only to 0.027 US$/kg SO 4 , assuming a bulk price of 35 US$/ton HCl. H 2 produced by water hydrolysis has also the added benefit of the O 2 produced, valued at 0.24 US$/kg H 2 in Chile ( Armijo and Philibert, 2020 ). Because hydrolysis generates 0.5 mol of O 2 /mol H 2 , and the proposed system requires 0.125 mol of O 2 /mol H 2 (reactions 1 and 2), the sale of the 75% surplus O 2 could generate savings of 0.18 US$/kg H 2 . Besides, the generated S 0 can be recovered and sold as fertilizer (fourth-quarter 2020 price of 69 US$/ton; U.S. Geological Survey, 2021 ) or used in bioleaching of ore, contributing to the sustainability of the treatment."
} | 9,613 |
38390176 | PMC10881560 | pmc | 6,082 | {
"abstract": "Escalating concern over global warming, which is mostly associated with deforestation, has led to the development of new classes of materials that can replace wood and better utilise natural resources. Presently, waste is a significant factor in recycling. In this regard, one of the leading contributors to waste is agricultural waste, which includes dried branches, leaves of trees, plants, and other organic materials. In the current study, waste from corn agriculture was utilised as a potential reinforcement for the fabrication of corn stalk-low density polyethylene (CS-LDPE) composites via an injection moulding technique at 170 °C. The different parameters were assessed to develop composites using CS, including physico-chemical, macromolecular, mineralogical, elemental, and morphological analysis. The amount of corn stalk (CS) was varied from 10 to 50 wt% with respect to the polymer. The mechanical, physical and thermal performance of the composites was examined. The density and water absorption of the composites were found to remain within the ranges of 1.00–1.11 g/cm 3 and 0.22–1.01 %, respectively, whereas these parameters increased as the proportion of CS increased. The thermal conductivity decreases with the addition of CS from 0.36964 ± 0.020 to 0.22388 ± 0.002 W/mK. It was observed that adding CS to the composites increased their tensile and flexural properties, but decreased their impact strength. The maximum flexural strength of 14.40 ± 1.558 MPa, flexural modulus of 752.53 ± 180.409 MPa, tensile strength of 10.49 ± 0.946 MPa and tensile modulus of 539.79 ± 91.044 MPa were observed with a 50 % CS content. The results suggest that these materials have considerable potential to serve as a cost-effective substitute for the conventional lignocellulosic fillers in the manufacturing of wood-plastic composites.",
"conclusion": "4 Conclusion It is extremely difficult to provide substitute materials for traditional timber-based materials for a variety of applications due to declining forest stocks and rising demand. In this case, the use of composite materials that maximise the use of waste materials is beneficial. The potential of using agricultural waste as a lignocellulosic reinforcement in polymer composite materials is immense. This approach will enhance sustainable manufacturing while also having a significant impact on waste management. In this study, waste from corn agriculture was utilised as a potential reinforcement for the fabrication of low density polyethylene-corn stalk (LDPE-CS) composites via an injection moulding technique at 170 °C. The technical strategy outlined in this study is intended to support a circular economy by providing sustainable replacements for synthetic fibres. The amount of corn stalk (CS) was varied from 10 to 50 wt% with respect to the polymer. The water absorption and density of the composites increased with increasing CS content and remained within the ranges of 1.00–1.11 g/cm 3 and 0.22–1.01 %, respectively. The impact strength and thermal conductivity decrease with the addition of CS. The flexural properties (flexural strength; flexural modulus) and tensile properties (tensile strength; tensile modulus) of the composites increased with the incorporation of CS. A maximum flexural strength of 14.40 ± 1.558 MPa, flexural modulus of 752.53 ± 180.409 MPa, tensile strength of 10.49 ± 0.946 MPa and tensile modulus of 539.79 ± 91.044 MPa were observed with a 50 % CS content. The results suggest that these materials have considerable potential to serve as a cost-effective substitute for the conventional lignocellulosic fillers in the manufacturing of wood-plastic composites.",
"introduction": "1 Introduction A significant proportion of India's economy is derived from agriculture. Different types of crops are cultivated in agro ecological region of India; along with that considerable amount of crop residues are generated that are left behind in fields after harvesting [ 1 ]. The types of waste materials that originate from the agricultural sector include straw and husk from rice and wheat, jute fibre, vegetables and food waste, sugarcane bagasse, groundnut shell, coconut husk, wood scrap from wooden mills, cotton and corn stalk [ 2 , 3 ]. Crop residues in the form of straw, husk and stalk are primarily associated for the use in mulching of soil, as a fodder for animals, bio-manure, raw material for thatching of rural housings and as a fuel for both residential and commercial purposes. Apart from these uses, large amounts of crop residues are burned and sometimes left as such in the field to decompose naturally. The inefficient burning produces harmful gases that pollutes the air and affects the natural habitat of flora and fauna [ [4] , [5] , [6] ]. India ranks 7th in production and 4th in area among nations that cultivate corn, accounting for around 4 % of global corn area and 2 % of total production [ 7 ]. After being harvested, corn produces many byproducts such as husks, leaves, stalks, and cobs, all of which are lignocellulosic materials rich in natural fibers [ 2 ]. Corn stalks also have pith and possess similar composition as hardwood; therefore, it can be utilised as potential filler for the development of wood plastic composites [ 8 , 9 ]. Stalk is a lignocellulosic material comprising prevalently of lignin, hemi-cellulose, and cellulose. The substantial amount of cellulose and hemi-cellulose contribute in production of bioethanol from corn stalk as they are easily convertible into fermentable sugars [ [10] , [11] , [12] , [13] ]. In most corn producing countries stalk has been used as a nutritive product for some animals, but due to its increased fibre and low protein content it is not feasibly digestible; however, certain physical, biological, and chemical treatments of corn stalk improve its feeding value [ [14] , [15] , [16] ]. In addition, corn stalk may likewise be used as a raw material for delivering worthwhile chemicals and bio-determined materials by means of the bio refinery technique. This approach would help in decreasing ecological contamination and creating new business openings in country areas [ 17 , 18 ]. Composites are mixtures of two or more different materials that possess superior properties compared to those of individual materials that participate in the fabrication of composites [ 19 , 20 ]. The investigations of corn stalk waste reinforced composites have pulled in due thought from academicians and industrialists for their unrivalled properties such as improved mechanical characteristics, reduced density, biodegradability and eco-accommodating materials [ [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] ] ( Table S1 ). Chen et al. [ 29 ] utilised corn stalk flour and hybridized it with sisal fibres for the fabrication of PVC based composites. They reported that due to hybridization, the tensile and flexural strengths increased by 49.5 % and 20 %, whereas the tensile and flexural moduli increased by 22.9 % and 60.5 %, respectively. A study by S. Liu et al. [ 30 ] used alkali treated corn stalk, chitosan and glutaraldehyde along with polypropylene matrix for manufacturing of wood plastic composites. In comparison to the composite reinforced by untreated corn stalk, cross-linking alterations led to 19.2 % more flexural strength and 8.3 % more flexural modulus. The water absorption of the WPCs also showed better performance in the case of the modified composites than in their unmodified counterparts. Ashori and Nourbakhsh [ 31 ], developed composites using bagasse, sunflower and corn stalk as reinforcement with polypropylene as the matrix along with a coupling agent. They found that the inclusion of a coupling agent enhanced the mechanical parameters, including the flexural, tensile, and impact strength. Furthermore, they mentioned better interconnectivity between the matrix and the reinforcement as a result of coupling agent addition. In this study, an injection moulding technique was used for the development of CS-LDPE composites with CS concentrations varied from 10 to 50 %. To develop composites using CS, various parameters such as physico-chemical, macromolecular, XRD, XRF, FTIR, and SEM of CS were evaluated. The influence of CS on the different properties of CS-LDPE composites was studied. The present study aimed to utilise waste generated from the agriculture sector by using it as a reinforcing material for the fabrication of low density polyethylene (LDPE) based composites via an injection moulding process for potential applications as electrical insulating materials, architectural & cladding wall panels and damping pads.",
"discussion": "3 Results and discussion 3.1 Corn stalk 3.1.1 Macromolecular analysis Table 3 lists the lignocellulosic makeup of CS. The three principal constituents of lignocellulosic material are cellulose, hemicellulose, and lignin. According to the literature review, cellulose was determined to be the primary component of CS. Because of its chemical structure, cellulose is intended to strengthen cell walls. Chemically speaking, cellulose is made up of linear glucose unit chains connected by −1,4-glycosidic linkages. Cellulose microfibrils are long, thread-like crystalline structures formed when the cellulose glucan chains connect through hydrogen bonds and van der Waals forces. CS had a 41.43 ± 0.709 % cellulose content with 26.10 ± 0.625 % hemicellulose, which are joined to cellulose by hydrogen bonds. Chemically speaking, mannose, 4- O -methyl- d -glucuronic acid, glucose, and the 5,6-carbon sugars xylose and arabinose are also members of the family of sugar polymers known as hemicelluloses. The structural features of the tree are caused by lignin, which is the second most common polymer on Earth after cellulose, and CS was found to possess 8.13 ± 0.667 % of it [ [33] , [34] , [35] , [36] , [37] , [38] , [39] ]. Table 3 Macromolecular composition of corn stalk. Table 3 Replication Cellulose (%) Hemicellulose (%) Lignin (%) R-1 42.20 26.80 7.47 R-2 40.80 25.90 8.13 R-3 41.30 25.60 8.80 Mean 41.43 26.10 8.13 SD 0.709 0.6245 0.667 3.1.2 Physico-chemical analysis The physico-chemical data of CS are reported in Table 4 . The results revealed that the bulk density, specific gravity and porosity of CS were 0.21 ± 0.030 g/cc, 0.96 ± 0.041 and 78.47 ± 3.22 %, respectively. The values of bulk density, specific gravity and porosity depend on the arrangement and size of particulates within the specified volume. The presence of minerals and the texture of the sample also affects these values. Due to the large quantity of organic matter, finely textured samples often have a low bulk density because the particles prefer to arrange into porous grains. A low bulk density and large pore space are the outcomes of this process. On the other hand, solid particles are closer together, and the bulk density is often higher in samples with low organic matter concentrations [ [40] , [41] , [42] , [43] , [44] , [45] ]. The CS has a low density, which presents a potential for its use as reinforcement in the fabrication of lightweight hybrid composite materials for a range of structural purposes. The specific gravity of CS is the dry bulk density with the voids removed in reference to the unit mass of water. The pH of the CS was determined to be 6.68 ± 0.005, while the electrical conductivity was found to be 1.14 ± 0.001 mS/cm. It may be the result of the biomass being shaken in the water bath during the digestion process, which led to the leaching of mobile ions. The pH and electrical conductivity are dependent on the mobility of mineral ions, presence of water soluble, organic and inorganic ions [ [46] , [47] , [48] , [49] , [50] ]. Table 4 Physico-chemical analysis of corn stalk. Table 4 Replication Specific Gravity pH Porosity (%) Bulk Density (g/cc) Electrical Conductivity (mS/cm) R-1 1.02 6.681 81.97 0.18 1.145 R-2 0.94 6.683 81.13 0.17 1.142 R-3 0.97 6.686 73.91 0.25 1.143 R-4 0.91 6.693 77.96 0.20 1.141 R-5 0.96 6.696 77.38 0.21 1.140 Mean 0.96 6.687 78.47 0.21 1.142 SD 0.041 0.005 3.221 0.030 0.001 3.1.3 FTIR analysis The graph for FTIR analysis of corn stalk is represented in Fig. 2 . Generally, the FTIR spectrum of any chemical compound consists of two primary regions, i.e., the fingerprint region, which is specific to each molecule and has a wavenumber between 400 and 1300 cm −1 , and the functional group region, which has a wavenumber between 1300 and 4000 cm −1 . The spectra of corn stalk is quite complicated in the fingerprint area and contain several bands that have been ascribed to the primary corn stalk constituents. It was observed from the graph that the broad absorption band at 3332.96 cm −1 corresponds to the O–H stretching vibration, and the small band at 2893.97 cm −1 represents the C–H stretching vibration. The small peaks observed at 1732.37, 1603.21, 1370.14 1240.84 and 1158.26 cm −1 represent C]O, C]C, C–H and C–O stretching vibrations, respectively. The sharp peak observed at 1032.20 cm −1 was due to the C–O stretching vibration, which is associated directly with the structural characteristics of the holocellulosic mass. The small peak observed at 897.29 cm −1 attributes to C– O –C stretching [ 42 , 45 , [51] , [52] , [53] ]. Fig. 2 FTIR spectrum of corn stalk. Fig. 2 3.1.4 XRF analysis The XRF analysis of CS is shown in Fig. 3 . To get duplicate findings, the test was run five times. The obtained results showed that potassium oxide (K 2 O) as the major component present in corn stalk with the amount of 44.28 %. It was observed that calcium oxide (CaO) was the second most obtained component present in corn stalk (12.79 %). Apart from potassium oxide (K 2 O) and calcium oxide (CaO), only silicon dioxide (SiO 2 , 11.00 %) is present above 10 %, while all the remaining components are present below 10 %. Most of the literature has shown SiO 2 as the major component, due to the preparation of samples for concentrating inorganic components [ 54 , 55 ]. Fig. 3 XRF of corn stalk. Fig. 3 3.1.5 XRD analysis Fig. 4 represents the XRD diffractogram of CS. From the figure, it can be observed that CS possessed both amorphous and crystalline regions. The sharp peak observed was typically due to the complex polymer cellulose, which contains amorphous and crystalline regions. Crystallinity can be defined as the ratio of the amount of crystalline area in a cellulose containing material to the total amount of cellulose sample including, both crystalline and amorphous areas. The XRD spectrum of CS showed an eminent peak at around 22 o while minor peak at around 15 o of crystalline and amorphous regions, respectively. The method suggested by Navarro-Pardo et al. [ 56 ] was adopted for calculating crystallinity index (equation (2) ). According to the XRD analysis, the crystallinity of CS is 30.02 %. (2) C r y s t a l l i n i t y I n d e x = A c A c + A a where, A c and A a represent areas under the crystalline and amorphous peaks, respectively. Fig. 4 XRD of corn stalk. Fig. 4 3.1.6 Scanning and optical microscopy Scanning electron microscopy (SEM) and optical microscopy (OM) were performed on corn stalk, and the results are presented in Fig. 5 . The SEM ( Fig. 5 a–d) and optical images ( Fig. 5 e–f) revealed that the fibre was cut during grinding, which resulted in an irregular geometry of the CS with sharp edges. As shown in the micrographs, the CS also has a coarse and rough surface, which is desirable for strengthening the mechanical coupling of CS and LDPE, improving the resilience of the interface between the two materials. Most of the CS had a diameter less than 1 mm and appeared to be a needle like structure. At higher magnifications, mechanical damage can be observed where the epidermal layer was destroyed and possibly amorphous contents such as hemicellulose and lignin were loosened during mechanical grinding. Fig. 5 SEM and optical images of the CS-LDPE composites. Fig. 5 3.2 CS-LDPE composites 3.2.1 Mechanical properties The mechanical properties, such as flexural strength, tensile strength, flexural modulus, and tensile modulus, of the CS-LDPE composites are reported in Table 2 (supplementary materials), and the effect of varying the CS content on the mechanical properties is shown in Fig. 6 (a–b). The results from various mechanical tests showed a consistent increase in the measured mechanical properties that increased in direct proportion to the quantity of reinforcement. The enhancement in the flexural properties of the composites is significant due to presence of the CS, which resists the bending force and results in better flexural strength than that of the pure polymer [ [57] , [58] , [59] ]. The rise in flexural strength with higher filler loading in CS-LDPE composites can be explained by the mechanism of flexural loading in three-point bending. When observing the cross-section of the composite, the area positioned above the neutral axis experiences compressive stress, while the area below undergoes tensile stress. Consequently, the altogether flexural behaviour of CS-LDPE composites is a result of both its tensile and compressive characteristics [ 58 ]. However, although the tensile properties also increase with increasing CS content, their presence did not significantly affect the tensile strength when comparisons were made between pure polymer and their reinforced counterparts. This difference is likely attributed to the small fibres (CS) that do not impart much resistance toward tensile force. Fig. 6 Mechanical properties of the CS-LDPE composites (a) flexural properties (b) tensile properties. Fig. 6 The flexural strength and flexural modulus of the CS-LDPE composites were recorded within the ranges of 4.71 ± 0.03–14.40 ± 1.55 MPa and 173.32 ± 47.26–752.53 ± 180.40 MPa, respectively. Similarly, the tensile strength and tensile modulus of the CS-LDPE composites were evaluated to be in the range of 7.034 ± 0.07–10.49 ± 0.94 MPa and 53.17 ± 4.03–539.79 ± 91.04 MPa, respectively. The predominant component of CS is crystalline cellulose, characterized by an aligned fibril structure and robust hydrogen bonding, imparting high stiffness. Consequently, incorporating CS into polymer-based composites enhances their stiffness. Although the amorphous polymer lignin in CS has little effect on the mechanical characteristics, it is essential for attaching cellulose fibrils and enabling effective stress transmission to cellulose units [ 60 ]. Thus, CS is added to LDPE to efficiently boost its stiffness without significantly increasing its density. Moreover, the mechanical grinding and superior dispersion of CS in the matrix contributed to a significant increase in the mechanical properties. A small and uniform particle size results in a wide surface area, leading to excellent interaction between reinforcement and the matrix, which is advantageous for transferring stress from the matrix to fillers [ [61] , [62] , [63] ]. Gomes et al. [ 64 ] conducted a study revealing a modulus of elasticity of approximately 138 MPa at a 15 % filler loading with recycled low-density polyethylene. In a separate investigation, Feng et al. [ 60 ] observed a tensile strength of approximately 10.5 MPa in corn stalk-PLA composites. Another research by A.B. Ismail et al. [ 65 ] reported a tensile strength of around 7 MPa and a tensile modulus of about 300 MPa in corn stalk-LDPE composites. The impact of CS concentration on the Impact strength of LDPE based composites is illustrated in Fig. 7 . The impact strength of the CS-LDPE composites ranged from 24.40 ± 2.63 to 3.90 ± 0.436 kJ/m 2 . The findings of the analysis suggest that with increasing CS loading, the impact strength decreases as the number of stress concentration site increases. The decrease in impact strength with the addition of CS can be explained by the fact that since the reinforcement is lignocellulosic and stiff, it contributes to the reduction in impact strength. Moreover, the presence of straw ends, defects and irregular shapes of CS also plays an important role in the initiation of cracks which leads to a decrease in impact strength [ 66 , 67 ]. Fig. 7 Impact strength of the CS-LDPE composites. Fig. 7 3.2.2 Physical properties The effect of the CS content on the density and water absorption of the CS-LDPE composites is shown in Fig. 8 . The obtained results showed linear a relationship between density and CS concentration. The density of the LDPE-based composites increased as the CS loading increased. However, there was no significant difference in the densities of the composites since the density of CS was low and the amount of polymer was constant throughout the fabrication of the composites. The density of the composites was found to be in the range of 1.00–1.11 g/cc. The water absorption values of the CS-LDPE composites ranged from 0.22 to 1.01 % after 24 h of immersion in water. The expected increase in water absorption of the composites was observed with the incorporation of CS owing to its hydrophilic nature. These results are probably due to water molecules forming hydrogen bonds with the free OH groups of CS and potential water molecule diffusion into the composite [ 64 , 68 , 69 ]. Fig. 8 Density and water absorption of CS-LDPE composites. Fig. 8 3.2.3 Thermal conductivity The thermal conductivity obtained for CS-LDPE composites are reported in Table 5 . The heat resistance of the composites was evaluated using a thermal conductivity study. The thermal conductivity of the CS-LDPE composites ranged from 0.36964 ± 0.020–0.22388 ± 0.002 W/mK. Ismail et al. [ 70 ] observed a comparable outcome in their study, reporting a result of 0.223 W/mK for a combination of 30 wt% rice straw and 70 wt% polypropylene. Similarly, in a distinct study by Oyekunle et al. [ 71 ], a thermal conductivity value of 0.217 W/mK for plywood was reported, aligning closely with the findings of the present study. However, the thermal conductivity values obtained for CS-LDPE composites are lower than those of various insulating materials. For instance, particle board exhibits a thermal conductivity of 0.4730 W/mK [ 71 ], sawdust composite has a value of 0.263 W/mK [ 72 ], wool-lime putty composite measures 0.266 W/mK [ 73 ], and bamboo fibre composite shows 0.340 W/mK [ 74 ]. Additionally, composites containing sawdust, petiole, straw, Argan shell, and palm fibres present thermal conductivity values ranging from 0.300 to 0.492 W/mK [ 75 ]. The outcome of the thermal conductivity analysis of the CS-LDPE composites showed that the thermal conductivity decreased with increasing CS content, indicating that the reinforcement had a lower thermal conductivity than the matrix since the core of the CS was porous, which facilitated air trapping and reduced the thermal conductivity [ 76 ]. The thermal conductivity of the composites is notably influenced by their density, lower densities led to increased porosity, resulting in higher thermal conductivity. The reduced thermal conductivity associated with higher material density is explained by the trapping of a significant volume of air within small pockets of the compact material. This confined air acts as an effective barrier to heat flow, reducing conductive heat transfer and improving the material's insulation capabilities. This outcome is consistent with the idea that there is an inverse connection between material density and airflow within the porous structure [ 77 , 78 ]. Table 5 Thermal conductivity of the CS-LDPE composites. Table 5 Replication LDPE CS-0 % LDPE CS-10 % LDPE CS-20 % LDPE CS-30 % LDPE CS-40 % LDPE CS-50 % 1 0.3786 0.2566 0.2510 0.2439 0.2341 0.2218 2 0.3888 0.2615 0.2552 0.2435 0.2347 0.2231 3 0.3504 0.2665 0.2519 0.2447 0.2306 0.2238 4 0.3845 0.267 0.2544 0.2429 0.2371 0.2246 5 0.3459 0.2669 0.2549 0.2446 0.2369 0.2261 Mean 0.3696 0.2637 0.2534 0.2439 0.2346 0.2238 SD 0.020 0.004 0.001 0.001 0.002 0.001 3.2.4 FTIR analysis of CS-LDPE composites The FTIR spectra of CS-LDPE composites are shown in Fig. 9 a. The peak in the region of 3420 cm −1 appeared on the addition of CS to LDPE owing to O–H vibrations [ 79 ], the intensity of which increases on addition of hydrophilic CS to the hydrophobic LDPE polymeric matrix. The characteristic bands observed at 2914 cm −1 and 2847 cm −1 are ascribed to symmetric and asymmetric vibration of methylene groups in LDPE [ 80 , 81 ]. The peak obtained at 1463 cm −1 attributed to scissoring vibrations of –CH 2 group [ [82] , [83] , [84] ]. The broad peak appeared after CS addition at 1034 cm −1 corresponds to the structural characteristics of the lignocellulosic mass [ 79 ]. The small band at 719 cm −1 represents phases of I β cellulose and lignin [ 65 , 85 , 86 ]. Even though the intensities of the peaks differed depending on the amount of CS present, the FTIR spectra of the CS-LDPE composites exhibited no noticeable alteration in relation to the changes in CS concentration. Fig. 9 FTIR (a) and XRD (b) of CS-LDPE composites. Fig. 9 3.2.5 XRD analysis of CS-LDPE composites The two distinct peaks were visible in the diffractogram of the LDPE ( Fig. 9 b), located at 21° and 23° 2θ values, which correspond to 110 and 200 reflections, respectively. In the same way, the diffractogram of the composite with 10–50 % CS showed two distinct peaks at 21° and 23° 2θ values, which correspond to 110 and 200 reflections, respectively [ 79 ]. When compared to the pristine LDPE, the diffractograms for the composites showed minor pattern variations. The d-spacing of CS-LDPE composites from 10 to 50 % CS concentration (4.2110 and 3.8113 Å, 4.1835 and 3.7889 Å, 4.1680 and 3.7857 Å, 4.1564 and 3.7667 Å, 4.1373 and 3.7542 Å) were found to be lower than that of the pristine LDPE (4.2189 and 3.8243 Å). Another finding was that the diffractograms of the composites showed less intensity of peaks when compared to the pristine LDPE. The composites estimated crystallinity index, which was determined by comparing the areas of the peaks attributed to the crystalline and amorphous phases, revealed that the pristine LDPE had a higher crystallinity index (39.83 %) than the CS-LDPE composites, which showed crystallinity indices of 38.69 %, 37.16 %, 36.93 %, 36.62 % and 35.21 % respectively. This suggests that the addition of fillers lowers the crystalline quality of the polymeric materials owing to restricted mobility of the molecular chains of polymer and presence of the amorphous hemicellulose, cellulose, and lignin in CS [ 83 , 87 ]. 3.2.6 Scanning electron microscopy The morphology of the CS-LDPE composites can be seen in Fig. 10 . As seen, CS is uniformly dispersed and effectively trapped in the matrix (LDPE), which allows for the proper distribution of CS within the whole matrix ( Fig. 10 e). Additionally, with a high polymer loading, the CS can completely encapsulate the plastic matrix, aiding in the dispersion of the CS with minimal or no defects ( Fig. 10 a). The stress distribution is enhanced by good dispersion, which improves the mechanical performance of the composites. The image appeared to have some number of voids and detached structures, which may be explained by the rupture and pulling out of the CS during the mechanical fracturing process ( Fig. 10 b–d). Fig. 10 SEM images of the CS-LDPE composites. Fig. 10"
} | 6,835 |
37542329 | PMC10403917 | pmc | 6,083 | {
"abstract": "Background Sesquiterpenes are designated as a large class of plant-derived natural active compounds, which have wide applications in industries of energy, food, cosmetics, medicine and agriculture. Neither plant extraction nor chemical synthesis can meet the massive market demands and sustainable development goals. Biosynthesis in microbial cell factories represents an eco-friendly and high-efficient way. Among several microorganisms, Saccharomyces cerevisiae exhibited the potential as a chassis for bioproduction of various sesquiterpenes due to its native mevalonate pathway. However, its inefficient nature limits biosynthesis of diverse sesquiterpenes at industrial grade. Results Herein, we exploited an artificial synthetic malonic acid-acetoacetyl-CoA (MAAC) metabolic pathway to switch central carbon metabolic flux for stable and efficient biosynthesis of sesquiterpene-based high-density biofuel precursor in S. cerevisiae . Through investigations at transcription and metabolism levels, we revealed that strains with rewired central metabolism can devote more sugars to β-caryophyllene production. By optimizing the MVA pathway, the yield of β-caryophyllene from YQ-4 was 25.8 mg/L, which was 3 times higher than that of the initial strain YQ-1. Strain YQ-7 was obtained by introducing malonic acid metabolic pathway. Combing the optimized flask fermentation process, the target production boosted by about 13-fold, to 328 mg/L compared to that in the strain YQ-4 without malonic acid metabolic pathway. Conclusion This designed MAAC pathway for sesquiterpene-based high-density biofuel precursor synthesis can provide an impressive cornerstone for achieving a sustainable production of renewable fuels. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-023-02370-8.",
"conclusion": "Conclusions In conclusion, we have demonstrated the potential of a synthetic MAAC pathway to synthesize sesquiterpene-based high-density biofuel precursor in S. cerevisiae in this study. The mechanism by with malonic acid induced the promotive biosynthesis of β-caryophyllene has been studied in both transcriptional and metabolic levels, which is strains with rewired central metabolism can devote more sugars to target production. Finally, the yeast strain accumulated β-caryophyllene at the highest levels in a flask fermentation under optimum conditions. To the authors’ best knowledge, this is the first report on the stable and cost-efficient bioproduction of β-caryophyllene via MAAC pathway with the highest titers obtained by yeast cell factories. This study broadens the application of MAAC pathway in the biosynthesis of high-value natural products, especially sesquiterpene-based high-density biofuel precursor β-caryophyllene.",
"introduction": "Introduction For decades, the excessive depletion of fossil fuels such as coal and petroleum caused atmospheric contamination, global warming and other serious issues, which impelling imperative efforts on exploiting renewable and sustainable alternatives to fossil fuels [ 1 ]. As the natural derivatives of isoprene, a variety of terpenoids have exhibited broad applications in industries of energy, food, cosmetics, medicine and agriculture [ 2 ]. Especially, several sesquiterpenes (C15) with the characteristics of compact structures and low hydrophilicity have been recognized as ideal renewable fuels, providing the attractive candidates for petroleum-independent energy [ 3 ]. For example, sesquiterpene β-caryophyllene has great potential to be used as the next-generation aircraft fuel component apart from its anti-inflammatory and antioxidant activities [ 4 , 5 ]. Although sesquiterpenes are the complex secondary metabolites of plants, the production mode by extracting from their natural sources often suffers from tedious procedures, extremely low yields and restricted source of materials [ 6 ]. As for chemical approach, resource limitations and toxicity of chemical raw materials do not meet the sustainable development goals [ 7 ]. Therefore, the development of alternative green approaches for the large-scale sustainable production of sesquiterpene have drawn global attentions. In recent years, microbial cell factories for sesquiterpene biosynthesis were regarded as an alternative to above methods due to their environmentally friendly and sustainable properties [ 8 ]. Until now, various synthetic pathways of sesquiterpene have been well-established in Saccharomyces cerevisiae [ 9 ]. In addition to efforts on rate-limiting enzymes engineering [ 10 ] and down-stream sesquiterpenes pathway engineering [ 10 ], strategies for enhancing acetyl-CoA supply have been also developed to meet the requirement for industrial production, including introducing heterologous routes related to acetyl-CoA synthesis [ 11 ] and compartmentalization engineering [ 12 ]. Malonic acid, or propanedioic acid, is a dicarboxylic acid with three carbons. It is well known as a competitive inhibitor of succinate dehydrogenase of TCA cycle in almost all organisms [ 13 ]. Nevertheless, it has been proved that malonic acid assimilation plays an essential role in symbiotic nitrogen metabolism [ 13 ]. Importantly, beside manufacturing applications, such as chemical synthesis of flavors, fragrances, and pharmaceuticals [ 14 ], malonic acid has also been used as a building block chemical to produce diverse high-value malonyl-CoA-derived compounds in microbial cell factories, such as fatty acids, polyketides [ 15 ] flavonoids [ 16 ] and even 3-hydroxypropionate (3-HP) [ 17 ] and glutaric acid [ 18 ]. In these pioneer works, malonyl-CoA can be generated from malonic acid through only two steps in E. coli , involving malonic acid transport protein and malonyl-CoA synthase, which is the shortest route relative to glucose metabolic pathway [ 15 – 18 ]. However, the employment of malonic acid for biosynthesis have been realized in E. coli rather than S. cerevisiae [ 15 – 18 ] . As the model eukaryotic microorganism, the introduction of malonic acid assimilation in S. cerevisiae microbial cell factory has great application prospects in the field of bioproduction [ 19 ]. Although remarkable achievements have been made in the biosynthesis of sesquiterpene in S. cerevisiae due to its original MVA pathways, carbon and energy metabolism are strictly regulated in cells, which greatly limit the highly efficient production of sesquiterpene [ 20 ]. To overcome this issue, here we firstly developed an alternative pathway termed malonic acid-acetoacetyl-CoA (MAAC) pathway that enabling to circumvent intrinsic inefficiencies of native central metabolism and thus enhancing the productivity of the targets in S. cerevisiae . Until now, few groups have attempted to produce β-caryophyllene in yeast [ 21 , 22 ]. However, the titers failed to meet demands of industrial production [ 21 , 22 ]. In our present work, to maintain stable yield of β-caryophyllene in S. cerevisiae , the genome sequence has been successfully reconstructed by introducing endogenous and heterogeneous genes. Carbon isotope labeling experiment and transcription analysis revealed that the rewired central carbon metabolic network efficiently promoted mevalonate pathway (MVA pathway) by increasing the availability of central metabolic intermediates acetyl-CoA and acetoacetyl-CoA (Fig. 1 ). Finally, β-caryophyllene production was significantly boosted after optimization of fermentation conditions, and these results further confirmed our proposed mechanisms to increase production mediated by MAAC pathway. Moreover, the recruitment of this route in biosynthesis of another sesquiterpene (C15) β-elemene also led to enhanced production and the titer of 984.36 ± 50.31 mg/L reached to the highest level ever reported. Therefore, this study expands the application fields of malonic acid assimilation pathway, from biosynthesis of malonyl-CoA-derived valuable compounds to biosynthesis of acetyl-CoA-derived natural high-value products. Fig. 1 Switching carbon metabolic flux for sesquiterpenes biosynthesis in S. cerevisiae . Native mentalism network is depicted with dark. Malonate can be transformed into acetoacetyl-CoA via synthetic MAAC pathway (red arrows). The engineered cytosolic MVA routes (green arrows) is responsible for β-caryophyllene formation. HXT7, high-affinity glucose transporter; HXK1, hexokinase; PDC, pyruvate decarboxylase; ADH2, ethanol dehydrogenase; ALD6, acetaldehyde dehydrogenase; ACS1, acetyl coenzyme A synthetase; ACC1, acetyl Coenzyme A carboxylase; Mae I, malonate transporter; MatB, malonyl-CoA synthase; Mat A, malonyl-CoA decarboxylase; ACCS, acetoacetyl-CoA synthase; ERG10, acetyl-CoA C-acetyltransferase; ERG13, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase; tHMGr, truncated HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, mevalonate pyrophosphate decarboxylase; IDI1, isopentenyl diphosphate isomerase; ERG20, farnesyl diphosphate synthase; QHS, β-caryophyllene synthase\n\nThe introduction of the MAAC pathway to enhance the production of sesquiterpenes Previous studies have been reported that acetyl-CoA was the crucial precursor for sesquiterpenes production [ 20 ]. Enhancements of acetyl-CoA supply for overproduction of sesquiterpenes have been accomplished by reconstructing metabolic pathways or tailoring cell organelles into microbial cell factories in previous studies [ 11 , 12 ]. Nevertheless, achieving economically viable titers is still a major roadblock in the process of reaching industrial production of high-value compounds. Aside from acetyl-CoA, acetoacetyl-CoA is a next vital precursor in MVA pathway, which can be obtained by the action of ERG10 from two molecules of acetyl-CoA. Thus, the increased supply of acetoacetyl-CoA was an alternative way to stimulate MVA pathway. A recently identified acetoacetyl-CoA synthase (ACCS) from Streptomyces sp. strain CL190, which catalyzes the condensation of malonyl-CoA and acetyl-CoA to generate acetoacetyl-CoA, has been proposed as a potential target to enhance production of acetoacetyl-CoA derived compounds. Unfortunately, production of farnesene did not benefit from ACCS expression [ 25 ]. It was speculated that malonyl-CoA represents a critical precursor for this pathway as well as for fatty acid synthesis and, therefore, its availability for the mevalonate pathway might be insufficient. Nevertheless, the blockage of fatty acid formation led to severe damage of cell growth [ 25 ]. Hence, we speculated the introduction of heterogeneous malonyl-CoA producing route might be a promising solution to balance cells growth and the biosynthesis of β-caryophyllene. In the present work, malonic acid assimilation pathway accompanied by ACCS or malonyl-CoA decarboxylase (MatA) were recruited to rewrite the central metabolism in two ways: (i) directly increasing supply of acetoacetyl-CoA or acetyl-CoA from non-fermentable carbon source malonic acid; (ii) reducing the carbon flow from acetyl-CoA to fatty acid biosynthesis by increasing the source of malonyl-CoA. Thus, the obvious reinforcement of sesquiterpenes production in current study was benefited from providing abundant malonyl-CoA for biosynthesis of β-caryophyllene and fatty acids. Shake-flask tests were carried out to determine the effects of the synthetic MAAC pathway in sesquiterpenes bioproduction. First of all, membrane transport limitations were addressed by incorporation of malonate transporters due to the toxicity of malonate to cells [ 26 ]. Until now, several transporters responsible for malonate transportation have been identified [ 17 ]. To achieve the superior activity in S. cerevisiae , we screened seven malonate transporters with reported activity on malonate transportation via expression from a plasmid pRS41H in CEN.PK2-1D, including Rl MatC from Rhizobium leguminosarum bv trifolii [ 13 ], Mae I from Schizosaccharomyces pombe [ 27 ], MdcF from Klebsiella pneumoniae [ 28 ], MadLM from Acinetobacter baylyi [ 29 ], tripartite ATP-independent periplasmic (TRAP) transporters from Rhodobacter capsulatus ( dct PQM) [ 30 ] and Sinorhizobium meliloti ( mat PQM) [ 31 ], respectively. The results showed that Mae I from S. pombe displayed the highest transport efficiency, which has reached to 25.67% (Fig. 3 A). Moreover, the amounts of malonate affected transport efficiency of Mae I. As depicted in Additional file 2 : Fig. S1, transport efficiency reached the highest value when the concentration of malonate was 60 mM. Thus, this content was used in following studies. Fig. 3 The construction of synthetic MAAC pathway for enhancing the production of β-caryophyllene. A Malonate transport efficiency by the action of malonic acid transport protein originated from various microorganisms. Recombinant strains were cultured in the YPD medium supplemented with 60 mM of malonate for 12 h. Then samples were taken for detection of malonic acid. B Gene expression levels of MatB from different origins in strains. Relative expression level was the intensity of MatB divided by the intensity of internal reference ALG9. C The amounts of malonyl-CoA in cells expressing Mae I and different sources of MatB after 36 h cultivation. D Relative β-caryophyllene production via four metabolic routes with malonate (blue bar) or not (yellow bar). The production of β-caryophyllene through MBC pathway in the presence of malonate was regarded as 100%. Data are presented here as mean values ± standard deviation (SD) calculated from n = 3 biological replicates. ** indicates significant pairwise differences between two bars, p < 0.01, *** indicates significant pairwise differences between two bars, p < 0.001 In the malonate metabolism pathway, the second key enzyme is malonyl-CoA synthase, which is responsible for the conversion of malonate to malonyl-CoA. To obtain the appropriate MatB in S. cerevisiae , we expressed MatB from three originates through plasmid pRS41H, including Rp MatB from Rhodopseudomonas palustris [ 32 ], Bj MatB from Bradyrhizobium japonicum [ 13 ] and At MatB from Arabidopsis thaliana [ 33 ]. As shown in Fig. 3 B, quantitative real-time PCR (qRT-PCR) analysis revealed that mRNA transcription levels of Bj MatB and Rp MatB genes was highly expressed in these strains over the At MatB strain. In vivo enzyme activity assay further determined the superior performance of Rp MatB in yeast (Fig. 3 C), which was corresponding to our previous results in bacteria [ 17 ]. Thus, Rp MatB was chosen for subsequent study based on the results of systematically determination at transcription and in vivo enzymatic activity levels. To produce our targets from malonate, the third stage is the synthesis of acetoacetyl-CoA from malonyl-CoA via two routes. The first pathway is only one step mediated by ACCS, and the second pathway is two steps mediated by MatA and ERG10. To evaluate the performance of these two pathways in production of β-caryophyllene, two engineered strains were constructed by introducing ACCS and MatA two genes into strain YQ-4 via plasmid harboring Mae I or Rp MatB encoding genes, respectively, generating engineered strains MBC and MBA. Compared to strain YQ-4, the level of β-caryophyllene in MBC and MBA strains lifted nearly 1- and 0.7-fold (Fig. 3 D), respectively. Besides, only overexpressing ACCS or MatA failed to improve the production of β-caryophyllene in the presence of malonate or not. All of these results demonstrated that the additional of malonic acid assimilation pathway can efficiently promote the production of our target.",
"discussion": "Discussion Acetyl-CoA is the basic precursor for sugar and fatty acid metabolisms, and even biosynthesis of isoprenoids. In the previous studies, excessive production of sesquiterpenes could be achieved by increasing the supply of acetyl-CoA [ 11 , 12 ]. Unfortunately, the titer of sesquiterpenes is still difficult to reach the level of industrial production. In our previous study, we adopted special malonic acid metabolic pathway to achieve the highly efficient supply of malonyl-CoA for producing 3-HP in E. coli [ 17 ]. Herein, expanding the applications of malonic acid assimilation strategy in biosynthesis of malonyl-CoA derived compounds to other valuable isoprenoids in yeast was attempted. To realize stable and controllable production of β-caryophyllene in S. cerevisiae CEN.PK2-1D, genome has been reconstructed by Cas9-based genome editing method. Initially, QHS from A. annua was introduced into S. cerevisiae CEN.PK2-1D due to its key role in synthesis process of β-caryophyllene. Subsequently, conventional strategy was carried out to improve the production of germacrene A, concentrating upon overexpressing heterogenous or native MVA pathway genes, such as tHMGr, ERG10, ERG20 and ERG13. Consistent with our expectations, the titer was lifted from 7.75 ± 0.01 mg/L to 25.80 ± 0.70 mg/L. To further strengthen the biosynthesis of isoprenoids in strain YQ-4, malonic acid assimilation pathway was introduced into this engineered S. cerevisiae . Malonate transporter and MatB were systematically determined at transcription and in vivo enzymatic activity levels, and Mae I and RpMatB were chosen as the highest efficient enzymes used to produce β-caryophyllene from malonate. In addition to these two steps, the enzymes responsible for conversion of malonyl-CoA to acetoacetyl-CoA were also optimized. The results revealed that ACCS-mediated route was much efficient than MatA-ERG10-mediated way. However, the performance of ACCS for bioproduction of farnesene was unsatisfactory probably due to the insufficiency of malonyl-CoA in cells [ 25 ]. Thus, the obvious reinforcement of isoprenoids production in current study was benefited from providing abundant malonyl-CoA for biosynthesis of germacrene A and fatty acids. Shake-flask tests were carried out to determine the effects of the synthetic MAAC pathway in isoprenoids bioproduction. To systematically uncover the underlying molecular mechanism, 13 C isotopic tracer analysis and quantitative RT-PCR assay were conducted to investigate the role of MAAC pathway in biosynthesis of germacrene A and fatty acids. Taken together, we speculated a tuned mechanism by with malonic acid induced the enhanced biosynthesis of β-caryophyllene. Initially, intracellular malonic acid transported by the action of Mae I may have inhibition influences on glucose transport and metabolism due to its toxicity to cell [ 28 ], resulting in limited β-caryophyllene synthesis. Then, with the conversion of malonic acid to malonyl-CoA catalyzed by MatB in cytoplasm, the inhibition was relieved and thus the glucose transportation and metabolism returned to normal level, leading to highly efficient production of β-caryophyllene. During which, the great majority of malonyl-CoA derived from malonate was recruited to produce fatty acids, and this precursor was sufficient for fatty acids synthesis process. Thus, more acetyl-CoA generated from glucose entered into MVA pathway to synthesize the product rather than fatty acids synthesis route. Although malonic acid failed to directly contribute the synthesis of the target, its assimilation in yeast efficiently rewrote the central carbon metabolism network, which was benefit to switch carbon flux towards β-caryophyllene from other metabolites. In addition to the pathway modification, fermentation condition was another vital point for high-level production of sesquiterpenes in this engineered yeast. It was reported that the isoform of QHS ( AaCPS1 ) contains highly conserved DDxxD and RxR motifs, responsible for Mg 2+ ion-substrate binding [ 24 ]. So, Mg 2+ was probably essential to catalytic activity of QHS due to 99% identity between AaCPS1 and QHS [ 24 ]. Results showed that the addition of Mg 2+ could strengthen the production of β-caryophyllene by improving the catalytic activity of QHS, which was consistent with previous efforts for producing other sesquiterpenes, such as patchoulol [ 37 ]. As well known, feeding modes is a very important factor for high-level of production. We found that the higher initial sugar supply only led to increase in biomass rather than production capability as described above. Thus, intermittent feeding strategy was attempted. After 144 h with 6 times supplement of glucose, the highest β-caryophyllene titer of 328.00 ± 13.44 mg/L was obtained, which was 13-fold higher than that of initial titer (25.8 ± 0.7 mg/L). The yield of YQ-7 from glucose to β-caryophyllene was 2.2 mg/g. The present titer of β-caryophyllene in engineered S. cerevisiae YQ-7 was 2- and 26-fold higher than the titers through adaptive evolution and engineering β-alanine metabolism in other S. cerevisiae , respectively [ 21 , 22 ]. As another sesquiterpenes, the biosynthesis of β-elemene has attracted great attentions [ 35 , 36 , 38 ]. Similarly, the improvement of the production of β-elemene by introducing MAAC metabolic pathway in yeast was also appropriate. The titer of 984.36 ± 50.31 mg/L in YQ-8 was 2- to 4-fold higher than the titers in S. cerevisiae in previous studies [ 35 , 36 ]. Moreover, the difference of consumed malonate amounts for two fermentation conditions implying the persistent effects mediated by MAAC pathway for production of our target. Therefore, these results confirmed our proposed mechanism described above. MAAC pathway can continually provide sufficient malonyl-CoA for native metabolism. So provided glucose concentrated on the biosynthesis of sesquiterpenes."
} | 5,405 |
22207865 | PMC3245565 | pmc | 6,085 | {
"abstract": "A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by 13 C-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO 2 was confirmed in a 13 C-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 × 10 9 cells cm −3 sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition–fluorescence in situ hybridization (CARD–FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD–FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea , but only small numbers of Eukarya . The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (δ - Proteobacteria , candidate division OP9, and Anaerolineaceae ) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae , which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.",
"conclusion": "Conclusion The first-time discovery of an AOM-influenced methane seep in the Indian Ocean was confirmed by the presence of dissolved methane as well as methane-dependent pro- and eukaryotic communities. Methane δ 13 C signatures indicate a microbial origin of methane. The released methane was oxidized by an active microbial community, sharing features with other seep communities. Albeit negligible in situ , it was also observed that higher hydrocarbons were converted to methane in vitro .",
"introduction": "Introduction Anaerobic oxidation of methane (AOM) has been described for decades, but became accepted as a key process in anaerobic carbon cycling only during the last 15 years (Davis and Yarbrough, 1966 ; Barnes and Goldberg, 1976 ; Reeburgh, 1976 , 2007 ; Knittel and Boetius, 2009 ). So far, it has been observed in many marine environments contributing significantly to carbon cycling in the sediments and the reduction of methane emissions. Continental margins and their forelands were examined in numerous biogeochemical studies (e.g., Bohrmann et al., 1998 ; Reed et al., 2002 ; Inagaki et al., 2006 ) and AOM was associated mainly to gas seeps and mud volcanoes (Aloisi et al., 2000 ; Joye et al., 2004 ; Valentine et al., 2005 ; Niemann et al., 2006b ; Lösekann et al., 2007 ). Such cold seep ecosystems, especially in the Eel River Basin (Orphan et al., 2002 ), the Hydrate Ridge (Bohrmann et al., 1998 ), the Black Sea (Michaelis et al., 2002 ), and the Gulf of Mexico (Roberts and Aharon, 1994 ), have been intensively studied regarding the geochemistry and microbiology of AOM. Within these AOM ecosystems, ANaerobic MEthanotrophs (ANME) populations are often the prevailing constituents and drive the biogeochemical processes. In sediment systems with diffusive methane fluxes, the distribution of ANME is restricted to the sulfate–methane transition zone (SMTZ), the only place where both methane and sulfate are available. The ANME populations and their sulfate-reducing partner bacteria are principally the same as those at cold seeps. However, cell numbers and activities of AOM-related populations are significantly lower (Knittel and Boetius, 2009 ). Although the importance of AOM in a global context has been widely recognized, the process is on a mechanistical and physiological level still not very well understood (Thauer, 2011 ). In the initial AOM reports, methanogenesis and sulfate reduction were believed to be mutually exclusive processes (Martens and Berner, 1974 ). However, AOM coincides with methanogenesis (Krüger et al., 2005 ; Niemann et al., 2006b ), and was therefore proposed to be reverse methanogenesis (Krüger et al., 2003 ; Scheller et al., 2010 ). So far, it was demonstrated that sulfate and nitrite reduction couple to AOM as a joint process of specialized methane oxidizers and sulfate- or nitrite-reducing microorganisms (Boetius et al., 2000 ; Raghoebarsing et al., 2006 ; Ettwig et al., 2010 ). Metal reduction coupled to AOM was also suggested (Beal et al., 2009 ). Phylogenetic analysis of AOM-sediments identified three novel groups of so-called ANME- Archaea , ANME-1, ANME-2, and ANME-3. These ANME are distantly related to cultivated methanogenic members from the orders Methanosarcinales and Methanomicrobiales (Orphan et al., 2002 ; Knittel et al., 2005 ; Niemann et al., 2006b ). FISH techniques showed that ANME occur in aggregates (Boetius et al., 2000 ; Michaelis et al., 2002 ; Knittel et al., 2005 ) with bacteria related to Desulfosarcina – Desulfococcus or Desulfobulbus . These findings suggest that AOM coupled to sulfate reduction is a syntrophic process, in which ANME convert methane to a metabolite which is used as electron donor by the sulfate-reducing bacterial partner. The Sumatra forearc is spatially remote from previous study sites. Therefore, our main objective on the R/V Sonne cruise SO189-2 into the Sumatra forearc basins was to detect and investigate methane seeps on the seafloor, and to describe the related geochemical and microbiological features in comparison to background sediments of nearby sites. After the successful first-time discovery of a methane-driven cold seep in this geographical region, we analyzed different biogeochemical proxies for AOM to determine related microbial activities. This was combined with molecular biological methods to study the involved microbial populations, to allow a comparison of the present results with those already published on other seep sites worldwide.",
"discussion": "Discussion For the first time, an active methane seep was discovered in the Indian Ocean. The discovered seep comprised highly active (stations 1 and 2) and less active or inactive (stations 3 and 4) AOM-influenced areas (Table 1 ; Figure 4 ). The highly active stations 1 and 2 were characterized by black sulfidic surface sediments, depleted sulfate, and high sulfide concentrations and light δ 13 C DIC values of the porewater, the presence of ANME-1 and ANME-2 representatives, as well as high cell and high copy numbers of 16S rRNA and functional genes related to AOM, methanogenesis, and sulfate reduction. A defined seep center of activity, like in mud volcanoes, was not discovered, the seep area was rather characterized by a patchy distribution of active spots. Carbonate- or sulfide-rich spots were distributed randomly over the surface. A reason for the patchiness might be tectonic activity. While some gas conduits might have been shut, other could have opened over time. An apparent feature of the active parts at the seep was the strong depletion of 13 C in DIC, which was also observed for TOC of the guts of the seep’s macro fauna. This confirms the importance of methane as carbon source for the benthos at this location. In addition, methanogenic activity was confirmed in sediment microcosms of the Simeulue seep area as well as in the Nias basin, where AOM activity was absent. Methanotrophy and sulfate reduction activities at the seep stations Methane is an indirect electron source for dissimilatory microbial sulfate reducers in the syntrophic process of AOM (Knittel and Boetius, 2009 ). The terminal reaction products are carbonate and sulfide. The produced sulfide in turn may be oxidized at the oxic/anoxic interface near the sediment surface. White, sometimes filamentous sulfide-oxidizing bacteria are typical indicators for this interface (Niemann et al., 2006b ). Areas covered with such white-colored microbial mats were discovered in the Simeulue basin and sampled in their proximity using a TV-guided grab (station 2, Figure 1 ). In contrast, microbial mats were absent near the station 1. Most likely, the white color of such mats is a result of intracellular sulfur inclusions as observed in Thioploca, Beggiatoa , or Thiomargarita aggregates, regularly found on the surface of sulfide-rich marine sediments (Gallardo, 1977 ; Jannasch et al., 1989 ; Schulz et al., 1999 ). At the Simeulue seep, methane was probably a carbon source for higher biota, as indicated by their 13 C-depleted carbon signatures. However, the δ 13 C (−31 to −45‰) values of the sampled crab guts indicate a mixed carbon source originating from AOM and water column carbon ( δ 13 C CH 4 −70.9 to 74.8‰, δ 13 C DIC −0.8 to −48.8‰, δ 13 C TOC −22.3 to 27.6‰). Heterotrophic processes as well as symbiosis between methanotrophic microorganisms and macrofauna are well described for several hot and cold deep marine vents (Childress et al., 1986 ; Duperron et al., 2005 ; Petersen and Dubilier, 2009 ). AOM was clearly a carbon donating process. It has been previously demonstrated that more than 99% of the methane in AOM systems is used for energy metabolism (Wegener et al., 2008 ). The oxidized carbon is than excreted as carbonate and probably reassimilated into biomass. The same seems likely for the Simeulue invertebrate community as previously shown for symbiotic CO 2 -fixing microorganisms and the gutless worm Olavius (Blazejak et al., 2005 ). Symbiosis of higher benthos and methanotrophic microorganisms is also often associated with aerobic methanotrophy (Childress et al., 1986 ; Duperron et al., 2005 ; Niemann et al., 2006b ; Petersen and Dubilier, 2009 ). However, the DGGE 16S rRNA gene analyses, focusing on dominant bands, did not reveal aerobic methane oxidizers in the seep sediments (Figures 5 and 6 ). The anoxic nature of the sediment was confirmed by porewater data, showing in particular high sulfide concentrations, reduced iron, and manganese as well as ammonium to be present (Figure 3 ). While sulfide concentrations increased downward into the sediment, sulfate decreased to micromolar concentrations. However, a clear SMTZ was not observed. Large amounts of dissolved gas with strong sulfidic odor evaporated from the sediment during sampling of the stations 1 and 2. Indicators for ongoing AOM at stations 1 and 2 were the low δ 13 C DIC values in the porewater apparently derived from 13 C-depleted methane (Tables 2 and 3 ). These values were comparable to other methane influenced seeps, as in the Gulf of Mexico (Coffin et al., 2008 ) or at the Hydrate Ridge (Valentine et al., 2005 ). Since ocean water δ 13 C DIC values are usually between 0 and −10‰ (Deuser et al., 1968 ), DIC at the Simeulue seep was obviously derived from the anaerobic oxidation of upward migrating methane (Figure 3 ). δ 13 C CH 4 values were below −70‰ at the stations 1 and 2 (Table 3 ). It is commonly agreed that biogenic methane exhibits δ 13 C CH 4 values below −70‰ (Whiticar et al., 1986 ). Hence, the observed methane at the Simeulue seep was likely biogenic methane as well. The AOM rates observed for the Simeulue seep area were slightly lower than maximum rates reported for other methane seeps, but higher than rates observed for mud volcanoes or sediments from various marine SMTZ (Knittel and Boetius, 2009 ). That methane rather than TOC was the carbon source for microorganisms is supported by low δ 13 C TOC values at station 1 and 2. These values were in a narrow range between −22.7 and −24.5‰ which are typical for marine cellular carbon (Deuser et al., 1968 ; Rice, 1993 ). These values contrast δ 13 C DIC values between −11.8 and −48.8‰ at the stations 1 and 2, most of them below −40.0‰ (Figure 3 ). Since the carbon isotopic composition of methane at both sites was below −70.0‰ (Table 3 ), it is obvious that AOM contributed to the DIC budget at these active AOM sites. The calculated SRR in the equilibrium zone, i.e., the zone of sulfur input from sulfate does not exceed the SRR, and was between 0.43 μmol cm −1 day −1 at 4 cmbsf and 0.56 μmol cm −1 day −1 at 8 cmbsf. The fluid flux was assumed to be 10 cm year −1 (Girguis et al., 2003 ) and the equilibrium was reached when the sum of sulfide and sulfate concentrations did not exceed the bottom water concentration of sulfate any more (27.79 mM, Figure 3 ). Moreover, the seep sediments were highly methane laden, as indicated by intensive gas emission during sampling. Huge discrepancies between sulfate reduction and AOM rates are usually observed only when methane plays a minor role in the investigated system (Niemann et al., 2006a ), which is not the case here. However, one may expect that there would have been a greater contribution of AOM derived carbon to TOC as observed, as methane was apparently a carbon source for the AOM performing microorganisms. Wegener et al. ( 2008 ) reported the assimilation of methane derived carbon into AOM performing microbial consortia of various geographic origins via CO 2 fixation. Furthermore, they could show that methane mostly serves microbial catabolism and to little extent microbial anabolism. This is also reflected in carbon stable isotopic signatures of DIC and TOC of the Simeulue seep, where stronger methane signals were detected in DIC and low δ 13 C TOC values do not support a significant impact of methane derived carbon to TOC (Table 3 ; Figure 3 ). On the other hand, ammonium concentrations decrease between 6 and 11 cmbsf at station 1. This could either be due to (i) heterotrophic TOC degradation for energy metabolism, (ii) hypothetical anaerobic ammonium oxidation with sulfate (Schrum et al., 2009 ), or (iii) ammonium uptake due to the increase of biomass upon AOM. TOC degradation would liberate ammonium and can be excluded (e.g., Wehrmann et al., 2011 ). Anaerobic ammonium oxidation with sulfate was suggested based on geochemical sediment profiles (Schrum et al., 2009 ; Wehrmann et al., 2011 ) but has not been convincingly demonstrated in vitro yet. However, it would have a stoichiometry of 1 mol sulfate reduced to 2.67 mol ammonium oxidized. From 4 to 6 cmbsf, the stoichiometry is 1–6.47 mol (Figure 3 ). That would indicate either an inefficient ammonium oxidation or a contribution of AOM to the ammonium decline. Given a one to one stoichiometry of sulfate driven AOM, the remaining 3.8 mol of sulfate could have been reduced by AOM. The measured AOM rate in the microcosm of station 1 was 0.57 μmol cm −3 day −1 (Table 3 ). During AOM, only 1% of the methane derived carbon is reassimilated into biomass (Wegener et al., 2008 ). That means 5.7 nmol carbon cm −3 day −1 were assimilated into biomass due to AOM. Given a relation of carbon to nitrogen in living organisms of 6 to 1, the ammonium uptake was 1.0 nmol nitrogen cm −3 day −1 . Given the ammonium influx from the water column was constant, the ammonium uptake exceeded the input between 4 and 6 cmbsf (Figure 3 ). Assuming a fluid flux of about 10 cm year −1 (Girguis et al., 2003 ), the ammonium uptake was about 20 μmol cm −3 day −1 . This exceeds the estimated ammonium uptake due to AOM by more than four orders of magnitude and therefore can not account for the decrease in this zone. As alternative explanation we propose ammonium oxidation with an unknown electron acceptor for this zone. Sulfate may be a candidate as suggested by Schrum et al. ( 2009 ). Since at station 4 AOM was not reflected by the porewater chemistry we postulate a former, recently ceased AOM activity, which has left its imprint in the carbon isotope signatures and ANME-related 16S rRNA gene biomarkers. Consequently, the δ 13 C TOC values below −27‰ are suggestive for a greater contribution of methane to TOC compared to DIC than observed for the stations 1 and 2 (Figure 3 ). AOM driven microbial activity, as indicated by an increase of ANME members with sediment depth (Figure 4 ), could have caused a stronger methane signal in TOC when methane was the prevailing carbon and energy source. Moreover, the impact of TOC of the water column is presumably much lower at a depth of 150–300 cmbsf than closer to the sediment surface. Methane was relatively enriched in 13 C at this station, which could also be well explained by AOM activity. Extremely low methane concentrations could have led to a less pronounced isotopic fractionation effect (Holler et al., 2009 ) – caused by rate limiting methane concentrations (Table 3 ). A number of in vitro experiments could confirm AOM as an important process at the discovered methane seep. Anaerobic methanotrophy was observed in initial and in transferred microcosms, as indicated by labeling experiments as well as by methane-dependent SRR (Table 3 ). That sulfate reduction was mainly driven by methane was further supported by the observation of comparable TOC values in the sediments of both, stations 1 and 2 as well as station 4 (Figure 3 ). If TOC degraders significantly accounted for sulfate reduction, depleted sulfate and high sulfide concentration would have been measured for station 4 as well, and not only for the stations 1 and 2 for which sulfate reduction via active AOM could be demonstrated. The higher rates in the first transferred microcosms indicated an enrichment of methanotrophs in these assays after incubation for more than 1 year as indicated by labeling experiments using 13 C-methane. Moreover, relatively high concentrations of reduced manganese were detected at station 2 (Figure 3 ). The presence of reduced manganese may be an indication for sulfide dependent or direct microbial manganese reduction. Sulfide dependent or direct microbial manganese reduction was probably a result of AOM activity at the Simeulue seep as suggested for the Eel river basin by Beal et al. ( 2009 ). Additionally, one sequence of the station 2 and four sequences of the station 3 were assigned to the Sh765B-TzT-29 cluster, a cluster that was initially believed to belong to the family Geobacteraceae and was first described by Geißler ( 2003 ). This cluster contains heavy metal associated Bacteria which where originally found in uranium mill tailings at Shiprock (NM, USA). A recent study described metal associated AOM consortia (Beal et al., 2009 ). Members of the Sh765B-TzT-29 cluster may also be metal associated at the Simeulue seep. Ádditionally, our culturing experiments with oxidized metals showed oxidation of methane, possibly associated to the Sh765B-TzT-29 cluster. Moreover, 16S rRNA gene sequences reported by Beal et al. ( 2009 ) were found in a close phylogenetic neighborhood of operational taxonomic units (OTUs) identified in the Simeulue seep (Figure 6 ). In conjunction with the presence of reduced iron and manganese in the porewater (Figure 3 ), it seems possible that metal reduction played a role as electron acceptor, besides sulfate, for AOM. Microbial community composition at the seep stations Anaerobic oxidation of methane supported a vital microbial community as demonstrated by 13 C-methane labeling experiments, rate measurements in vitro , rate estimations in situ , the presence of ANME-related mcrA genes, and an active archaeal and bacterial community at stations 1 and 2 (Figure 4 ). Many archaeal sequences obtained from the Simeulue seep stations 1 to 4, were distributed over the ANME-1 and -2 clusters (Figure 7 ). OTUs from the stations 1 and 2 were assigned to ANME-1b and ANME-2b clusters. Also at the station 3, ANME-2a/c members were identified. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis at both AOM-stations and the station 3. The occurrence of Crenarchaeota at a methane seep is not unusual and has been reported for other sites (Knittel et al., 2005 ; Knittel and Boetius, 2009 ). Most bacterial groups belonged to the family Anaerolineaceae (phylum Chloroflexi ), the candidate division OP9 (Webster et al., 2004 ) and the class δ- Proteobacteria . These groups have been described to be dominant in marine sediments (Teske, 2006 ; Blazejak and Schippers, 2010 ). Sequences affiliated to the family Anaerolineaceae were derived from stations 1 to 4. The nearest cultured genus Leptolinea (distance matrix: 84.5% identity with the nearest Leptolinea member) has been described as saccharolytic, including pectin and cellulose degrading species (Yamada et al., 2006 ; Ishii et al., 2008 ). In these studies, members of the genus Leptolinea were not able to reduce sulfate or other sulfur species (Yamada et al., 2006 ). Leptolinea and Levilinea species largely comprise taxa discovered in anaerobic waste water sludge (Rivière et al., 2009 ), indicating the possibility of active heterotrophic processes at the AOM seep. However, sequences from the Simeulue seep were closer related to sequences which could not be assigned to these genera (Figure 6 ). Three sequences of the stations 1 and 2 were closely related to the genus Desulfococcus of the family Desulfobacteraceae , which is in good agreement with previously described AOM sites (Knittel and Boetius, 2009 ). At the stations 1 and 2 (only data for station 1 are shown in Figure 4 ), mcrA genes encoding for the enzyme methyl-CoM-reductase of the anaerobic methanotrophic ANME-2 group dominated over ANME-1. ANME-1 mcrA genes prevailed only at the surface of the station 2. The Methanosarcina (“methanogenic”) type of the mcrA gene was detected throughout the whole seep area, while it was completely absent in other sediments of the Sumatra forearc (Schippers et al., 2010 ). The simultaneous occurrence of both, methane production and oxidation in the seep area, underscores the important role of the methane cycle for this system. The dsrA gene, an indicator for the presence of sulfate reducers, was found in slightly higher gene copy numbers at the station 1–3 (Figure 4 ). Catalyzed reporter deposition–fluorescence in situ hybridization cell counts, assessing the active community, indicated that living Bacteria were present with at least two orders of magnitude more cells than Archaea at the station 2. At the station 1, active bacterial and archaeal cells were distributed equally. In contrast, results obtained by qPCR, targeting also inactive microorganisms, indicate a dominance of Bacteria over Archaea by one order of magnitude. The total cell numbers of 10 7 to 10 9 cells cm −3 at the Simeulue seep were quite similar to those at sites of the Sumatra forearc basins not influenced by methane seepage (Schippers et al., 2010 ). These are also comparable to cell numbers reported for the arctic, methane emitting, Haakon Mosby mud volcano, hosting mainly aerobic methylotrophs (Niemann et al., 2006b ). Also the decrease of cell numbers with sediment depth was similar compared to this mud volcano. CARD–FISH cell counts, and qPCR measurements showed the presence of Bacteria and Archaea , but only small numbers of Eukarya . This observation is in agreement with previous marine sediment studies (Schippers and Nerretin, 2006 ; Schippers et al., 2010 ). Methanogenesis as a concomitant process Another goal of this research was to demonstrate methanogenic hydrocarbon degradation (Zengler et al., 1999 ; Head et al., 2003 ). From a geological point of view, the Sumatra forearc seems a promising location for hydrocarbon generation in the deep subsurface, a potential source for upward migrating of complex hydrocarbons. Hence, all stations were screened for such processes. Hydrocarbon-dependent methanogenesis was observed in microcosms of the Simeulue seep and the Nias basin. After a first transfer of sediment microcosms of five stations (Table 2 ), three stations showed sustained methanogenesis in the presence of higher hydrocarbons. Only one of these stations was located in the seep area (station 6), the other two in the Nias basin. The rates estimated in the initial setups as well as in the first transfers, were in the same range compared to a hydrocarbon adapted community of contaminated harbor mud in the North Sea (Siegert et al., 2011 ). Methanogenesis was absent in microcosms without added substrates. Possibly, the presence of 28 mM sulfate and other electron acceptors inhibited methanogenesis from TOC, but not from higher hydrocarbons. In contrast, it seems likely that the addition of higher hydrocarbons stimulated activity of TOC and hydrocarbon utilizing microorganisms. Positive controls containing the substrates TMA or methanol with 28 mM sulfate confirmed that methanogenic activity in spite of present sulfate. Methanogenesis from these substrates evolved rapidly within the first weeks of incubation and was in the same order with the AOM rates. Desulfobacteraceae species may be indicative for AOM consortia (Knittel and Boetius, 2009 ), but one member of this family is the hexadecane degrader Desulfococcus oleovorans strain Hxd3 (Aeckersberg et al., 1991 ; So et al., 2003 ). Hence, the occurrence of this family suggests the presence of consortia capable of anaerobic degradation of higher hydrocarbons. The finding of other closely related hydrocarbon seep associated sequences, e.g., from mud volcanoes or contaminated sites, confirmed this (Figure 6 ). However, hydrocarbons are abundant substances in nature and our culturing experiments show that the presence of hydrocarbon degraders does not necessarily depend on the presence of hydrocarbons in higher concentrations. In summary, hydrocarbon utilizing methanogenic communities were present in the Sumatra forearc sediments irrespective of methane seepage, and their presence does not infer that higher hydrocarbons played a significant role in the carbon cycle. Nonetheless, this is the first report of stable microcosms of hydrocarbon-dependent methanogenic microbial communities from the deep ocean."
} | 6,538 |
39404665 | PMC7616573 | pmc | 6,086 | {
"abstract": "This perspective article focuses on the innovative field of materials-based bacterial engineering, highlighting interdisciplinary research that employs material science to study, augment, and exploit the attributes of living bacteria. By utilizing exogenous abiotic material interfaces, researchers can engineer bacteria to perform new functions, such as enhanced bioelectric capabilities and improved photosynthetic efficiency. Additionally, materials can modulate bacterial communities and transform bacteria into biohybrid microrobots, offering promising solutions for sustainable energy production, environmental remediation, and medical applications. Finally, the perspective discusses a general paradigm for engineering bacteria through the materials-driven modulation of their transmembrane potential. This parameter regulates their ion channel activity and ultimately their bioenergetics, suggesting that controlling it could allow scientists to hack the bioelectric language bacteria use for communication, task execution, and environmental response. Graphical abstract",
"conclusion": "Conclusion In conclusion, the dynamic interplay between bacteria and material interfaces opens new avenues for bioengineering, with the potential to revolutionize multiple fields, including energy production, environmental remediation, and medical applications. The general rationale is to couple and harmonize existing biological attributes, which are defined by evolution, with exogenous ad hoc functionalities borrowed from materials. Ultimately, this materials-driven paradigm has the potential to be applied broadly to any bacterial species, whereas genetic rewriting can suffer from a limited range of applicability. In addition to standard applications of engineered microbes in energy harvesting/storage and environmental remediation, I want to highlight two novel research routes that may stem from this field, namely, i. achieving control over the morphogenesis, composition, and functionality of microbial communities and ecosystems and ii. hacking the bioelectric code of bacteria. The development of the former research field has important implications for the study and engineering of microbiomes, such as the human gut or epidermis microbiomes. The latter would allow us to explore the uncharted territory of bacterial bioelectricity, which, in my view, represents the software that regulates their activity.",
"introduction": "Introduction Bacteria play a pivotal role in diverse ecosystems, ranging from soil and oceans to the Earth’s atmosphere and, critically, the human body. As essential drivers of nutrient cycling, symbiotic relationships, and ecological balance, bacteria profoundly influence the health and sustainability of various environments. From the production of oxygen through photosynthesis to the colonization of diverse niches within the human body, including the gastrointestinal tract, [ 1 ] epidermis, and even the once-thought-aseptic environment of the brain, [ 2 ] bacteria have played a profound role in the genesis and evolution of humans. Such continuous interaction of bacteria with diverse environments, as well as their strong adaptability, have conferred them with unique attributes finely tuned by evolution. Here I list some relevant examples, alongside with the related applications: Photosynthetic bacteria harness the power of sunlight to produce energy, a process with applications in sustainable energy production, such as bio-solar cells and artificial photosynthesis [ 3 ] ; Electrogenic bacteria possess the ability to promote redox reactions through extracellular electron transfer pathways, offering potential applications in microbial fuel cells and electrochemical cells for electricity generation and environmental remediation; Bacteria can exhibit motility, [ 4 , 5 ] allowing them to navigate diverse environments. This feature has important implications for micromotor technology [ 6 , 7 ] and targeted drug delivery systems in body locations that are difficult to reach or navigate due to the low Reynold’s number, [ 8 ] \n i.e., the gastrointestinal tract or tumors [ 9 , 10 ] ; Bacteria can assemble into microbial communities with sophisticated decision-making processes, [ 11 – 13 ] influencing the health of crops and humans and offering opportunities for the production of engineered living materials with tailored properties. In the last decades, the relatively new field of synthetic biology seeks to exploit the unique properties of these microorganisms for transformative applications. [ 14 ] This represents a state-of-the-art technique for serving to this purpose. [ 15 , 16 ] By rewiring genetic circuits and metabolic pathways, synthetic biologists can tailor bacteria to extend and augment their living attributes, such as their ability to harvest light energy [ 17 ] or undergo extracellular electron transfer, [ 18 ] as well as to encode microbes with programmable functionalities, such as the responsivity to optical and chemical stimuli. [ 19 ] While a significant portion of synthetic biology focuses on the engineering of genetic components, it is important to acknowledge that material approaches can also have profound effects on bacterial gene expression and protein function. The integration of abiotic materials with bacterial systems can influence genetic pathways in ways that complement traditional synthetic biology techniques. For instance, recent developments have demonstrated the potential of electronically controlling gene expression using materials-based systems. [ 20 ] Focusing on materials as a “stand-alone” tool for controlling the fate of eukaryotic cells and organisms, our research community has recently turned its attention to the use of exogenous materials for this purpose. The general approach involves utilizing exogenous nano/molecular materials: their nanometer-scale dimensionality is essential for establishing reliable abiotic interfaces with biological molecules and cells. Additionally, the artificial functionalities of these materials, such as optical and electronic responsiveness, enable the equipping of living matter with extra mechanisms that complement existing ones. [ 21 – 23 ] This relatively new paradigm has now extended into microbes. [ 14 ] In the past, the interaction between materials and microbes has been extensively studied and exploited for bacterial eradication and the treatment of related infections, [ 24 – 26 ] as well as for the development of bacterial sensors to hinder the spread of pathogenic bacteria. [ 27 – 32 ] However, the current approach involves viewing bacteria as engineerable materials, with the goal of achieving new functionalities or enhancing existing ones through material interfaces, [ 33 ] for instance, by injecting energy to bacteria to allow them to operate away from equilibrium. [ 34 ] Beyond investigating the fascinating world of bacterial electrophysiology and complex communication, [ 13 , 35 , 36 ] this approach can offer unprecedented opportunities to address pressing societal challenges. These include the development of new and intrinsically sustainable energy harvesting systems based on bio-mimetic photosynthetic hybrids, [ 37 – 40 ] microbial fuel cells for power production, [ 41 ] bio-electrosynthesis platforms where microbes produce desired chemicals, [ 42 ] and novel engineered living systems that combine features of materials with living attributes that have been refined by evolution, such as motility and adaptation (Fig. 1 ). [ 43 – 45 ] Figure 1 Schematic summarizing the general contents in this perspective. In this perspective article, I explore the multifaceted landscape of material-driven strategies in bacterial engineering. For clarity, in this article, I use this term to refer to the fine manipulation of bacterial functions driven by the interface with exogenous abiotic materials, with the general aim of augmenting existing features or conferring new ones. Additionally, note that the scope of this manuscript is not to provide a comprehensive overview of the extensive literature on microbial engineering. Instead, I aim to highlight recent interdisciplinary research efforts that share common materials-driven approaches, serving the purpose of bacterial engineering. For each specific topic I will show only a few recent case studies that are summarized in Table I . First, I will give a brief overview on the use of carbon-based materials for the enhancement of bioelectric capabilities of bacteria to undergo extracellular electron transfer (Section “ Introduction ”), as well as to enhance the ability of photosynthetic microbes to harvest sunlight (Section “ Engineering bioelectricity generation through material interfaces ”). In Section “ Advancements in photosynthetic biohybrid systems ”, I will present recent studies on the use of functional materials to modulate bacterial communities, while in the Section “ Microbial communities and materials ” I will briefly talk about the fascinating opportunities arising from the use of bacteria as biohybrid microrobots. In Section “ Conclusion ”, I will discuss a general paradigm for engineering bacteria, consisting of the materials-driven modulation of their transmembrane potential. This is essentially the parameter that regulates their ion channel activity and ultimately their bioenergetics, implying that achieving control over it would permit hacking of the bioelectric language that bacteria use for communicating, carrying out tasks, and responding to their environment. [ 46 ] Table I Summary of the case studies reported in this perspective paper, categorized by bacteria, materials, and the resulting functions from the material interfacing. Bacteria Materials Applications/functions Shewanella oneidensis Graphene Conjugated polyelectrolyte Microbial fuel/electrochemical cells Improve charge collection Improve charge extraction Synechocystis E. coli Carbon nanotubes Conjugated polymers Indium Tin Oxide NPs Hybrid photosynthetic systems Enhance photo-exoelectrogenicity Improve photocurrent collection Gut microbiota bacteria E. coli A. xylinum / B. subtilis Artificial soil Hydroxyapatite Hydrogel ink Modulation of microbial communities Enhancement of gut bacterial diversity Modulation of mineralization Degradation of pollutants E. coli Magnetic NPs Bacterial microrobots Cancer cells ablation Drug delivery B. subtilis Silicon nanowires Gold NPs Azobenzenes Modulation of bacterial bioelectricity Photomodulation of calcium signaling Electromodulation of cell potential Photomodulation of cell potential"
} | 2,645 |
21678949 | null | s2 | 6,087 | {
"abstract": "Bacteria have developed a cell-to-cell communication system, termed quorum sensing (QS), which allows for the population-dependent coordination of their behavior via the exchange of chemical signals. Autoinducer-2 (AI-2), a class of QS signals derived from 4,5-dihydroxy-2,3-pentandione (DPD), has been revealed as a universal signaling molecule in a variety of bacterial species. In spite of considerable interest, the study of putative AI-2 based QS systems remains a challenging topic in part due to the rapid interconversion between the linear and cyclic forms of DPD. Herein, we report the design and development of efficient syntheses of carbocyclic analogues of DPD, which are locked in the cyclic form. The synthetic analogues were evaluated for the modulation of AI-2-based QS in Vibrio harveyi and Salmonella typhimurium. No agonists were uncovered in either V. harveyi or S. typhimurium assay, whereas weak to moderate antagonists were found against V. harveyi. On the basis of NMR analyses and DFT calculations, the heterocyclic oxygen atom within DPD appears necessary to promote hydration at the C3 position of cyclic DPD to afford the active tetrahydroxy species. These results also shed light on the interaction between the heterocyclic oxygen atom and receptor proteins as well as the importance of the linear form and dynamic equilibrium of DPD as crucial requirements for activation of AI-2 based QS circuits."
} | 357 |
31910889 | PMC6947943 | pmc | 6,090 | {
"abstract": "Background The impact of human activities on the environmental resistome has been documented in many studies, but there remains the controversial question of whether the increased antibiotic resistance observed in anthropogenically impacted environments is just a result of contamination by resistant fecal microbes or is mediated by indigenous environmental organisms. Here, to determine exactly how anthropogenic influences shape the environmental resistome, we resolved the microbiome, resistome, and mobilome of the planktonic microbial communities along a single river, the Han, which spans a gradient of human activities. Results The bloom of antibiotic resistance genes (ARGs) was evident in the downstream regions and distinct successional dynamics of the river resistome occurred across the spatial continuum. We identified a number of widespread ARG sequences shared between the river, human gut, and pathogenic bacteria. These human-related ARGs were largely associated with mobile genetic elements rather than particular gut taxa and mainly responsible for anthropogenically driven bloom of the downstream river resistome. Furthermore, both sequence- and phenotype-based analyses revealed environmental relatives of clinically important proteobacteria as major carriers of these ARGs. Conclusions Our results demonstrate a more nuanced view of the impact of anthropogenic activities on the river resistome: fecal contamination is present and allows the transmission of ARGs to the environmental resistome, but these mobile genes rather than resistant fecal bacteria proliferate in environmental relatives of their original hosts. \n Video abstract.",
"conclusion": "Conclusions In this study, we evaluated a river model ecosystem exhibiting characteristic resistome dynamics driven by anthropogenic impacts. Snapshots taken from the river continuum under a gradient of anthropogenic pressures provided novel insights into how human activities shape the environmental resistome. Our results demonstrate that fecal contamination could be responsible for the introduction of ARGs into the anthropogenically impacted river resistome, but human-related mobile resistance genes rather than resistant fecal bacteria proliferate in environmental relatives of clinically important proteobacteria.",
"discussion": "Discussion Several studies have reported a positive correlation between the abundance of ARGs in the environment and anthropogenic activities [ 27 – 29 ]. In principle, the effect of anthropogenic activities on the environmental resistome could be mediated by two types of processes: input of human-related ARGs into the environment and selection pressure for the carriage of ARGs [ 30 ]. The latter process is often hypothesized to promote the spread of mobile ARGs among bacterial communities in the environment. Evaluation of these ecological and evolutionary scenarios in environmental settings has been difficult due to the absence of appropriate data supporting these hypotheses. In the present study, we employed a river model ecosystem exhibiting a resistome succession driven by a gradient of anthropogenic activities at highly populated downstream regions, and we evaluated dynamics in human-related ARGs that occur over the course of such a transition in the river ecosystem. A recent study based on metagenomics analysis of a human fecal indicator bacteriophage showed that quantitative dynamics of ARGs from anthropogenically impacted environmental samples could be primarily explained by human fecal pollution, implying that input events rather than on-site selection pressures play a critical role in anthropogenic effects on the environmental resistome [ 19 ]. Other studies have shown that increases in ARGs in anthropogenically impacted rivers are accompanied by concomitant increases in pathogenic bacteria and human gut microbiome-associated sequences [ 18 ]. The present study also showed that both fecal phage and representative fecal bacteria increased in the downstream regions, although these fecal factors were not enough to fully explain the ARG bloom in the downstream regions. Accordingly, this raises the question of how much of the increase in ARGs in anthropogenically polluted environments is contributed by ARGs introduced from human-related bacteria and how much is contributed by ARGs indigenous to the environment. In the river system studied here, a large proportion of ARGs was shared with the human gut or pathogen resistomes. These human-related ARGs increased more steeply in the downstream regions than the other ARGs found in the river metagenomes, highlighting the fact that these genes are the major components of anthropogenically driven bloom of the river resistome. Notably, river-specific ARGs also increased 4.6-fold in the downstream regions, similar to human-related SCGs, suggesting that fecal input is not the major reason for the ARG bloom. The association of ARGs with MGEs is known to facilitate the spread of ARGs within and between environments through HGT [ 30 ]. Therefore, the localization of ARGs on MGEs has a critical influence on the fate of ARGs in the environment [ 24 ]. Many studies have reported elevations in MGE abundance in environments with anthropogenic influences [ 19 , 29 ]. Class 1 integrons are the most well-established indicator of such an influence [ 24 , 27 ]. However, the hypothesis that the spread of mobile ARGs is especially relevant to anthropogenically influenced environments has not been systematically examined. In the present study, we observed an increase in MGE abundance and the frequent genetic linkage of ARGs and MGEs in downstream regions. Furthermore, our results suggest that mobile ARGs play a dominant role in the anthropogenic transition of the river resistome. ARG sequences shared among river, human gut, and pathogens were frequently found in MGE contexts and were observed across a broader phylogeny of bacterial genomes. These human-related ARGs were not concomitantly detected with core phylogenetic marker genes. Our results suggest that ARGs rather than ARB are selected and these ARGs are mobilized and transferred laterally between different taxa in the downstream regions under high anthropogenic influences. The identification of bacteria carrying ARGs or displaying resistance phenotypes is critical for monitoring, risk assessment, and management of the environmental resistome. The taxonomy-resolved structure of the environmental resistome has mostly been evaluated in culture-based studies, which are able to assess resistance phenotypes and genetic determinants of isolates [ 14 , 20 ]. Recently, several studies have demonstrated host-tracking of the environmental resistome based on the taxonomic classification of metagenomic contigs harboring ARGs [ 31 ]. Although metagenomics approaches have advantages over culture-dependent approaches in terms of elucidating comprehensive and unbiased resistome profiles, especially for complex environmental communities, they are limited in terms of providing accurate taxonomic information and solid phenotypic evidence. In the present study, we harnessed both culture-dependent and -independent approaches to generate an integrative picture of ARG host ranges and the phenotype-level resistome. Using a metagenomics approach, we found that the overall host range of the river resistome was limited to a small number of branches across the bacterial phylogeny. Four proteobacterial families were the major hosts of ARGs, and their contigs showed a higher ARG density in downstream regions. It is noteworthy that all four families playing a major role in the downstream resistome encompass clinically important human pathogens. Analysis of resistance phenotypes in over 1500 ARB isolates complemented the taxonomic prediction of ARG hosts based on metagenomic contigs. A recent study showed that ARG profiles derived from functional metagenomics screening and the resistance phenotypes of coliform isolates from a sewage system were correlated [ 32 ]. Likewise, in some bacterial taxa whose ARG contents differed considerably between upstream and downstream regions based on metagenomics analysis, such as Acinetobacter , Aeromonas , Enterobacteriaceae , and Pseudomonas , we observed significantly different resistance phenotypes between upstream and downstream isolates. Such differences were not observed among isolates belonging to other taxa. In particular, most of these isolates from downstream regions exhibited decreased susceptibility against various classes of antibiotics. This observation was consistent with the increased human-related ARGs from these four proteobacterial lineage at the downstream regions, suggesting that human-related mobile ARGs are horizontally transferred to the environmental relatives of their original hosts and proliferate in the environment. Collectively, our results from both metagenomics-based analysis of ARGs and phenotypic analysis of ARB isolates showed similar trends, validating our robust characterization of river resistome dynamics driven by anthropogenic activities. Addressing what evolutionary mechanisms at the individual genome and pan-genome levels lead to the spread of these particular mobile ARGs and how the environmental resistome in turn influences the resistome in clinical settings are the next steps for better understanding the global dissemination of antibiotic resistance."
} | 2,349 |
38684659 | PMC11058813 | pmc | 6,091 | {
"abstract": "Agriculture contributes to a decline in local species diversity and to above- and below-ground biotic homogenization. Here, we conduct a continental survey using 1185 soil samples and compare microbial communities from natural ecosystems (forest, grassland, and wetland) with converted agricultural land. We combine our continental survey results with a global meta-analysis of available sequencing data that cover more than 2400 samples across six continents. Our combined results demonstrate that land conversion to agricultural land results in taxonomic and functional homogenization of soil bacteria, mainly driven by the increase in the geographic ranges of taxa in croplands. We find that 20% of phylotypes are decreased and 23% are increased by land conversion, with croplands enriched in Chloroflexi, Gemmatimonadota, Planctomycetota, Myxcoccota and Latescibacterota . Although there is no significant difference in functional composition between natural ecosystems and agricultural land, functional genes involved in nitrogen fixation, phosphorus mineralization and transportation are depleted in cropland. Our results provide a global insight into the consequences of land-use change on soil microbial taxonomic and functional diversity.",
"introduction": "Introduction Due to increasing human activities and agricultural intensification, an emerging body of research suggests that ecological communities are undergoing fundamental changes across various spatial dimensions 1 . Most studies investigating the consequences of land-use changes and agricultural expansion on ecological communities have focused on local species diversity 2 – 4 due to its ease of measurement and monitoring 5 . Such studies are relevant to highlight the loss of global biodiversity loss and species extinction 6 – 8 . However, in addition to reducing local species diversity, agricultural conversion also caused biotic homogenization at larger spatial scales 9 – 11 , posing a significant concern for ecosystem services and conservation. Biotic homogenization refers to the increase in taxonomic or functional similarities among ecological communities distributed spatially over time 12 . Biotic homogenization can be quantified by a decrease in β -diversity, e.g., a decrease in compositional dissimilarity between sites. Biotic homogenization can occur due to the establishment of exotic species (increasing similarity between communities), the loss of native species specific for a limited set of locations (reducing similarity) or most likely a combination of both 13 , 14 . Indeed, both natural pressures and anthropogenic activities, such as climate change, agricultural expansion, urbanization and habitat homogenization, could cause biotic homogenization 9 , 15 – 19 . So far, the impact of land use and agricultural conversion on biotic homogenization mainly focused on aboveground habitats 18 , with limited attention given to belowground communities. The information about agriculture-induced biotic homogenization of belowground communities is essential for regional biodiversity planning and conservation purposes. Land-use change and agricultural conversion can alter community assembly processes, community composition and species diversity concurrently 4 , 20 – 22 . These changes are underpinned by species extinction, colonization and uneven shifts in relative abundance among different geographic regions. Intense agriculture can contribute to soil compaction, salinization, acidification, metal accumulation, organic matter loss and nutrient imbalance 23 . These related environmental stressors generally induce structural shifts in microbial taxonomic and functional composition 24 , 25 , such as the retention of acid-tolerant taxa and the loss of specific functional traits for pathogen suppression or crop fitness 26 , 27 . Consequently, these shifts create ecological feedbacks that further influences soil functions critical for maintaining soil health and agricultural productivity. Despite numerous studies examining the responses of microbiome composition and function to agricultural conversion 28 – 31 , these observations are predominantly site-specific and limited to a local scale 3 , making it challenging to infer whether shifts in specific microbial taxa are relevant to the diverse range of soils worldwide 32 . Currently, we still lack a generalizable and consistent understanding of how soil microbial taxonomic and functional profiles respond to agricultural conversion and which microbial lineages and functions are mostly impacted across a wide range of soil and climate types. This knowledge gap hinders our comprehensive understanding of the global decline in biodiversity and associated ecosystem functions. In the present study, we address two major questions: (1) whether agricultural effects lead to taxonomic and functional biotic homogenization of soil microbiomes at large spatial scales? (2) how land-use changes alter soil microbial community composition and functions across a wide range of soil and climate types, and which microbial lineages and functions most strongly impacted? We combined a continental soil survey and a global-scale meta-analysis to address these questions. For the first question, we conducted a continental soil survey of 1185 samples from agricultural fields and the adjacent natural ecosystems (covering forest, grassland, wetland; Fig. 1c ) across China to provide large-scale evidence of agriculture-induced taxonomic and functional homogenization of soil microbiomes. To gain a global perspective on agricultural-induced biotic homogenization, and to complement the continental scale soil survey, we also collected 16S rRNA amplicon-based sequencing data from soil samples of global agricultural-natural ecosystem pairs from all available gene banks (Fig. 1a ). We hypothesized that agricultural conversion causes taxonomic and functional homogenization of soil microbiomes. For the second question, we used the continental survey dataset to explore general patterns of soil microbiome taxonomic and functional responses to agricultural conversion across a wide range of soil and climate types. We also determined how these responses vary among ecosystem types and different microbial lineages. Our results demonstrate that land-use change for agricultural purposes reduces taxonomic diversity in soil bacterial communities. Fig. 1 Taxonomic and functional homogenization of microbial communities in response to agricultural impacts at global and continental scale. a Data distribution of one-to-one correspondence of 2403 sequencing data between cropland and natural ecosystems across countries and continents. b Response of community similarity to agricultural conversion. The each bars represent the mean ± standard errors (SE). Asterisks indicate significant difference (wilcoxon test, *** p < 0.001). Non-metric multidimensional scaling of Bray–Curtis distances showing community dissimilarities between cropland and forest. Cropland, 1033 samples; Forest, 1370 samples. c Map showing 44 regions covering croplands and adjacent natural ecosystems. Typical terrestrial ecosystems, including croplands, forests, grasslands and wetlands. d , h Effect sizes of natural ecosystems impacts on β -diversity in taxonomic composition ( d ) and functional composition annotated with KEGG ( h ) relative to croplands. The estimated effect sizes are regression coefficients based on the linear models. Data are presented as mean ± s.e.m. of the estimated effect sizes. Sample size is showed by number of data pairs for each group. Statistical significance is based on F -test; *** p < 0.001, ** p < 0.01, * p < 0.05. e – g Principal coordinate analyses of Bray–Curtis distances showing dissimilarities among taxonomic composition between croplands and natural ecosystems, including forests ( e ), grasslands ( f ), and wetlands ( g ). A total of 303 forest-cropland pairs ( e ), 275 grassland-cropland pairs ( f ), and 278 wetland-cropland pairs ( g ) were compared. Each point indicates a site, and error bars around the means represent standard error of samples in given a site. i – k Principal coordinate analyses of Bray–Curtis distances showing dissimilarities among functional composition annotated with KEGG between croplands and natural ecosystems, including forests ( i ), grasslands ( j ), and wetlands ( k ). A total of 10 forest-cropland pairs ( i ), 10 grassland-cropland pairs ( j ), and 10 wetland-cropland pairs ( k ) were compared. Communities differed among ecosystem types using PERMANOVA: *** p < 0.001, ** p < 0.01, * p < 0.05.",
"discussion": "Discussion Agricultural land-use change has exerted profound effects on above- and belowground biodiversity 2 , 44 , and the effects are likely to accelerate in the coming decades 18 . While a number of studies showed that agricultural conversion led to biotic homogenization of aboveground communities, still very few studies investigated the belowground consequences. In the present study, we summarized the generalized effects of land-use conversion on belowground microbial communities and functions, encompassing multiple ecosystems. Our study provides large-scale evidence of taxonomic and, to a lesser degree, functional homogenization of soil microbiomes following agricultural conversion in terrestrial ecosystems at global and continental scales. The taxonomic variation across sites (Beta-diversity) was significantly lower in croplands than in grasslands, wetlands, and forests, pointing to biotic homogenization in croplands. Although land-use changes and agricultural conversion have been proven to be major drivers of biodiversity loss 45 , 46 , positive impacts of agriculture on biodiversity have been observed at regional and local scales in some studies 47 – 49 . One facet of these trends is that although local or alpha diversity may increase, this is typically at the expense of beta diversity 12 . Previous studies have demonstrated that increases in local land use intensity led to biotic homogenization of microbial, plant, and animal groups both above- and below-ground 4 , 25 . Biotic homogenization is largely independent of changes in alpha diversity; land use intensity reduced local alpha-diversity in aboveground groups, but increased the α-diversity in belowground groups 4 . Our study further extends these earlier observations at a continental and global scale and now provides widespread evidence that agricultural conversion results in biotic homogenization of the soil microbiome. Although taxonomic homogenization in cropland versus natural ecosystems was stronger and more significant in many cases, we observed very important microbial functional shifts under croplands, including functional homogenization 15 , 50 . This was evident when we calculated the beta-diversity across sites based on functional gene composition. Since the functional components of biodiversity are fundamental parts of ecosystem functions and services 51 , 52 , functional homogenization is the most direct evidence for the potential loss of ecosystem functions caused by agricultural conversion 16 , 53 . Our findings extend taxonomic-level results in Amazonian Forest 21 and European grasslands 4 that focus on the impact of agricultural management on belowground taxonomic homogenization in local-scale, to the large-scale functional homogenization. Overall, our study provides a comprehensive insight that agricultural land-use change cause biotic homogenization in taxonomic and functional composition, and suggests halting reclamation and developing ecological restoration for cropland to conserve landscape-scale biodiversity and ecosystem service provision 53 , 54 . Biotic homogenization in response to agricultural impacts is a multifaceted process that involves considering the invasion and extinction of species, as well as the heterogeneity of landscapes. In agricultural systems, it is generally believed that the biomes are a subset of the regional species pool, which is composed of surrounding natural ecosystems 55 . This highlights the selective effects of agricultural conversion, which could cause pressure and force on soil communities from natural ecosystems. For example, the destruction of soil structure and aggregates, as well as alterations and homogenization in soil environmental conditions caused by agricultural conversion can result in the trait-based filtering out of certain species, leading to the loss of existing species and the dominance of microorganisms that are better adapted to agricultural management. Moreover, geographic range size is a major determinant of species’ extinction risk, and rare species therefore are vulnerable to land use change and are at greater risk of extinction 25 . The establishment of agricultural systems through intensive management can facilitate the spread of colonizing species that are abundant and prevalent due to the characteristics of broad environmental adaptation, while rare or specialized species may decrease in their abundance and occupancy over time 56 , 57 , which led to a homogenization of community composition across space. Land-use change is proposed to affect turnover in community composition via its effect on stress tolerance, resource acquisition, and dispersal ability. Stronger stress-tolerant, broader resource-flexibility cosmopolitan species with unlimited dispersal capacity are more stable to land-use change because of increasing adaptive potential and/or extensive ability to exploit soil resource availability 58 , 59 . Frequently disturbed soil environments can promote the gains and proliferation of novel species and the gradual replacement of locally distinct communities by cosmopolitan communities via altered competitive and coexistence dynamics 60 , homogenizing assemblage composition. On the other hand, the influence of agricultural conversion on biotic homogenization might be attributed to the reduction in environmental heterogeneity in monoculture-dominated landscapes 61 . Landscape heterogeneity is central to the spatial organization of ecological communities 62 . Variations in vegetation structural and soil conditions influence beta diversity and turnover of soil fauna, bacteria, and fungi. Monoculture-dominated croplands have lower environmental heterogeneity compared with vegetation structural complexity in natural ecosystems, where heterogeneous habitats contribute to increased beta diversity across spatial scales. Our findings, supported by the estimation of ecological processes based on β NTI and RC Bray (Fig. 4b and Supplementary Fig. 10 ), illustrate that the role of homogeneous selection was stronger for community assembly in croplands, suggesting the consequence of agricultural conversion on homogeneous abiotic and biotic conditions across space. The impact of agriculture on biotic homogenization might vary at different scales. In contrast to our results, a regional survey on the conversion of steppe to cropland demonstrated that agriculture increased spatial heterogeneity of soil functional genes 29 . The lower functional turnover in steppe may be attributable to stable and similar soil environments across the region. Diverse in local but functionally homogeneous sward in regional natural steppe ecosystem exerts a stabilizing effect on the soil environment and soil ecosystem processes, reducing the impact of spatial and temporal variation in climate, soil texture and topography 29 . Differently, agricultural management such as seasonal planting, crop types, and fallow cycles actually contribute to greater temporal and spatial variability that selects for greater heterogeneity across the region. Given the complexity of the soil environment, more attention needs to be paid to the biotic homogenization caused by agricultural conversion of the soil microbiome at various spatial scales. Our results showed that land use change had a greater impact on taxonomic composition than on functional composition, highlighting the functional redundancy of soil microbiomes 35 , 63 , 64 . Soil microorganisms represent the most biologically and phylogenetically diverse community on Earth 65 . Although the taxonomic composition of soil microbiome varies tremendously across soil, microbial gene composition or functional capacity remains highly conserved 63 , 66 , with lots of phylogenetically unrelated taxa carrying similar genes and performing similar functions 38 . For example, lignin substrate can be degraded by gram-negative bacteria Comamonadaceae and Caulobacteraceae , and the genus Asticcacaulis and Caulobacter (members of Caulobacteraceae ) could degrade both hemicellulose and cellulose and all three lignocellulosic polymers, respectively 67 . Numerous microorganisms with the ability to participate in carbon degradation can coexist on the surface of plant residues 68 . Agricultural conversion, however, had minimal impact on overall carbon degradation and fixation, but did reduce nitrogen fixation and phosphorus mineralization and transportation potential (Fig. 3 ), suggesting the functional redundancy for carbon metabolism in soils. The fact that the potential for nitrogen fixation and phosphorus mineralization is reduced, indicates that croplands rely less on these processes due to the breakdown of nutrient cycling plant-microbial symbioses under agricultural fertilization. Taken together, our results indicated that agricultural land-use change significantly altered microbial taxonomic composition while the gene content remains relatively conserved, especially in relation to carbon metabolism. More realistic functional gene expression studies the functional divergences, redundancies, and complementarities in the different land use scenarios, e.g. metatranscriptomics 69 or quantitative stable-isotope probing (qSIP) 70 , that correlate with the observed taxonomic shifts after agricultural conversion, needs to be further revealed in the future. Changes in soil microbial communities across space are often strongly correlated with differences in soil abiotic and biotic conditions 47 . Similar to previous study 71 , we observed soil pH is a major driver of the diversity and composition of soil bacterial communities across land-use types. More importantly, we found that fungal communities, particularly pathogens and saprotrophs, were strongly associated with changes in soil bacterial communities. Interactions between fungi and bacteria could partly drive the bacterial community shifts along a steep gradient of fungal community change 72 , 73 . For example, manipulating fungal richness can immediately mediate assembly processes of bacterial community 74 . The fungal hyphae could provide soil bacteria with ecological opportunities in severely carbon-limited soils by releasing carbonaceous compounds and providing a colonizable surface for the creation of new bacterial niches 72 , 73 . In addition to the effect of external conditions (e.g., biotic interactions and abiotic environmental conditions), our results also emphasize the important roles of microbial traits in regulating the response of microbial composition to agricultural conversion. The dormancy potential strategy changed from sporulation and toxin–antitoxin systems to resuscitation-promoting factors 42 . The sporulation trait affects species composition, with the abundance of phyla Firmicutes and Actinobacteria with spore-forming ability 75 increasing in croplands. The impact of regional species pools on cropland bacterial diversity is modulated by sporulation trait 55 . Many taxa with spore-forming ability had a higher species pool effect, indicating their survival and competitive advantage under environmental stress, as well as their retention during land use changes or their greater likelihood of spreading from natural ecosystems due to their adaptive capabilities. Our findings provide a valuable insight for predicting ecological consequences of land-use change and agricultural management. The links between microbial composition and ecosystem function suggest that biotic homogenization have previously unrecognized and negative consequences for agricultural sustainability and service. Although the functional redundancy with C metabolism of soil microbiomes supports the stability and resilience of ecosystem functioning in response to perturbations 63 , increased agricultural intensification gives rise to large uncertainty in predicting the loss of ecosystem function. It is also important to note the ways observations at different spatial scales can impact the interpretation of broad soil microbiome responses. Although our study covered a global scale, study sites and sequencing data were not evenly distributed. Most observations focus on forest-cropland ecosystem contrasts and are subject to methodological limitations arising from comparisons of sequencing methods and sampling schemes. Overall, our study suggests that biotic homogenization of the belowground microbiome across large spatial scale should be taken into account when evaluating the sustainability and soil health of agricultural management practices."
} | 5,299 |
36307673 | PMC9614761 | pmc | 6,094 | {
"abstract": "Ocean warming has both direct physiological and indirect ecological consequences for marine organisms. Sessile animals may be particularly vulnerable to anomalous warming given constraints in food acquisition and reproduction imposed by sessility. In temperate reef ecosystems, sessile suspension feeding invertebrates provide food for an array of mobile species and act as a critical trophic link between the plankton and the benthos. Using 14 years of seasonal benthic community data across five coastal reefs, we evaluated how communities of sessile invertebrates in southern California kelp forests responded to the “Blob”, a period of anomalously high temperatures and low phytoplankton production. We show that this event had prolonged consequences for kelp forest ecosystems. Changes to community structure, including species invasions, have persisted six years post-Blob, suggesting that a climate-driven shift in California kelp forests is underway.",
"introduction": "Introduction As oceans warm, marine communities are exposed to novel and stressful abiotic and biotic conditions. Such is the case during acute periods of anomalous ocean warming—marine heatwaves—which are predicted to increase in severity and frequency over the next century due to anthropogenic climate change 1 , 2 . Recent marine heatwaves have caused extensive ecological and socioeconomic damage to communities in tropical and temperate ecosystems worldwide 3 , including severe coral bleaching 4 and massive die offs of seagrass and kelp 5 , 6 . Marine heatwaves directly stress organisms via the lethal and sublethal physiological consequences of increased temperatures 4 , 7 , but also have more indirect ecological consequences. For example, prolonged warming and resultant ocean stratification can retard upwelling of deep, nutrient-rich water to sunlit surface waters 8 , 9 . Temperate coastal ecosystems rely on upwelled nitrogen to fuel primary production by phytoplankton that sustain food webs 10 , 11 and macrophytes such as seagrasses and kelps that provide critical habitat for diverse marine communities 12 , 13 . Consequently, the extremely productive coastal ecosystems in upwelling regions may be particularly susceptible to ecological damage by marine heatwaves. Sessile invertebrates provide food for an array of mobile invertebrate and vertebrate predators on shallow temperate rocky reefs, acting as a critical trophic link between the plankton and the benthos through suspension feeding 10 , 14 . Their dependence on plankton may make this diverse group of primary space holders particularly vulnerable to the effects of marine heatwaves and continued ocean warming 15 , and a recent meta-analysis found that sessile species are generally more susceptible to the adverse effects of heatwaves than mobile species 16 . Most of the research done to date on the effects of marine heatwaves on sessile invertebrates has focused on hermatypic corals in the tropics, while on temperate reefs many studies have emphasized habitat-forming macroalgae and seagrasses rather than sessile invertebrates 4 – 6 , 15 – 17 . Given their ecological importance in temperate systems and vulnerability to environmental change, a more comprehensive assessment of the effects of anomalous warming on these diverse communities seems warranted. In 2014–2015, a persistent high-pressure zone pinned warm water masses along North America’s west coast; the resulting extreme heatwave was unprecedented in recorded history and became known as the “Blob” 9 , 18 , 19 . Positive temperature anomalies were accompanied by negative chlorophyll- a anomalies across the Southern California Bight in the winter and spring of 2014 and 2015 19 , due to deepening of the thermocline and nitracline and increased stratification that limited nutrient enrichment of the photic zone 9 . Here we use 14 years of seasonal data to evaluate the resilience of communities of sessile suspension-feeding invertebrates on reefs in the Santa Barbara Channel to the anomalous ocean conditions associated with the Blob. Sessile invertebrate abundance and species richness declined significantly, and species composition changed during the heatwave in response to depressed phytoplankton abundance that compounded the detrimental effects of warming. These changes, including greater abundance of nonindigenous and warmer-water species, have persisted 6 years post-Blob, suggesting that a prolonged climate-driven shift in the community structure of California kelp forests is underway.",
"discussion": "Results and discussion The Santa Barbara Coastal Long Term Ecological Research program has monitored benthic communities in five kelp forests seasonally since 2008 using fixed transect diver surveys, and moored sensors at each reef have recorded bottom temperatures every 15 min. Blob-associated positive bottom temperature anomalies began in winter 2014 and persisted through autumn 2016 (Fig. 1a ) 18 . Peak temperature anomalies occurred during the summer and autumn of 2014 and 2015 (Fig. 1a ), and the average temperature anomaly in autumn 2015 was +3.1 °C, equivalent to an average daily temperature of 19.6 °C. In 2014 and 2015, 91 and 69% of autumn days, respectively, were classified as heatwave days as defined by Hobday et al. 20 . Seasonal chlorophyll- a concentration, a proxy for phytoplankton abundance, was obtained from satellite imagery at each of the five reefs over the 14-year period. The average chlorophyll- a concentration was anomalously low throughout the warming period, and exceptionally low during the springs of 2014 and 2015 (Fig. 1a ), when upwelling-driven nutrient enrichment typically supports dense phytoplankton blooms. Fig. 1 Average seasonal bottom temperature anomaly, chlorophyll- a concentration anomaly, and percent cover and species richness of sessile invertebrates across five sites. The Blob, an anomalous warming period from spring of 2014 to winter of 2016, is highlighted in gray, coincident with ( a ) positive temperature anomalies (°C; solid line), negative chlorophyll- a anomalies (mg/m 3 ; dashed line), and declines in ( b ) invertebrate cover (solid line) and species richness (number of unique species/taxa/80 contact points; dashed line). Seasons are denoted by Sp (Spring), Su (Summer), A (Autumn) and W (Winter). Mean sessile invertebrate cover averaged across all sites declined 71% during the Blob, reaching a 14-year minimum of 7% in autumn of 2015 (Fig. 1b and Supplementary Fig. 1 ). Species richness declined 69% during the same period (Fig. 1b and Supplementary Fig. 1 ). The responses of invertebrates to warming were not consistent across time even though the duration and intensity of warming was similar in 2014 and 2015, suggesting that extended periods of elevated seawater temperature were not solely responsible for the most severe loss of invertebrates. For ectotherms, increases in ambient seawater temperature should be met with increases in metabolic rate and food requirements to sustain metabolism 21 . Because of their sedentary lifestyle, sessile invertebrates cannot actively forage for food or seek spatial refuge from thermal extremes, and limitations in their planktonic food supply can result in metabolic stress over extended periods 22 , 23 . Anomalously low chlorophyll- a concentrations during the Blob (Fig. 1a ), particularly in the spring of 2015, indicated that food limitation was a likely driver of invertebrate decline. Results from piecewise structural equation modeling (Fig. 2 ) that incorporated biological interactions with space competitors (understory macroalgae), predators (sea urchins), and foundation species (giant kelp) showed that the severity of warming had both a direct and indirect effect on the sessile invertebrate community. The proportion of heatwave days was a direct negative predictor of sessile invertebrate cover (−0.11) and species richness (−0.21). The proportion of heatwave days was an even stronger negative predictor of chlorophyll- a concentration (−0.26), yielding negative indirect effects on invertebrate cover (−0.07) and species richness (−0.05) due to the positive influence of chlorophyll- a concentration on sessile invertebrate cover (+0.26) and richness (+0.20). Fig. 2 Piecewise structural equation modeling (SEM) for sessile invertebrate cover and species richness. Arrows indicate directionality of effects on ( a ) invertebrate cover and ( b ) species richness. Red arrows show negative relationships; black arrows show positive relationships. R 2 values are conditional R 2 . Arrow widths are proportional to effect sizes as measured by standardized regression coefficients (shown next to arrows). *** p < 0.001, ** p < 0.01, * p < 0.05. Insignificant pathways are not included. Consequences of heatwaves on benthic community structure can be difficult to predict if community-level interactions exacerbate or ameliorate stressful conditions 24 , 25 . The biomass of giant kelp, Macrocystis pyrifera , an important foundation species on temperate reefs, was not strongly impacted by the Blob in the Santa Barbara Channel (Fig. 2 ) 18 , 26 , but experienced warming-induced declines at its equatorward range edge 26 , 27 . Given the strong positive relationship between giant kelp biomass and sessile invertebrate cover (+0.40) and species richness (+0.46), future declines in giant kelp during warming events, as has been seen in other kelp forest systems 5 , 17 , will likely contribute to sessile invertebrate declines. Similarly, loss of kelp canopy has been shown to increase understory algal cover and biomass 28 , 29 , a strong negative predictor of invertebrate cover (−0.45) and species richness (−0.32), apparently due to increased competition between understory algae and invertebrates for space. Invertebrate phyla did not respond equally to the Blob; some phyla experienced severe declines during the warming event while others resisted change. Bryozoans, sponges, annelids, and ascidians were particularly hard-hit and declined to their lowest percent cover during the Blob (Fig. 3 , Supplementary Fig. 2 ). For example, in autumn 2015 bryozoans and sponges were not recorded at any site while ascidians were recorded at only one site in very low abundance (i.e. ~1% cover). The decline in the percent cover of annelids began prior to the Blob, making it difficult to attribute their low abundance in 2015 solely to warming. Bryozoans and annelids rapidly recovered following the Blob, and by 2017 their percent cover approached or surpassed pre-heatwave levels. By contrast, the recovery of sponges was considerably slower, with pre-Blob levels not evident until 2020 (Fig. 3 , Supplementary Fig. 2 ). Mollusks were relatively unaffected by the heatwave, although their highest cover was observed in the years following the Blob, suggesting that they may have benefited from delayed indirect positive effects of warming (Fig. 3 , Supplementary Fig. 2 , Supplementary Fig. 4 ). Fig. 3 Average annual percent cover of invertebrate phyla. Gray shading indicates the Blob period of 2014– 2015 when bryozoan, ascidian, sponge (Porifera) and annelid cover declined dramatically. Although the percent cover of most sessile invertebrate phyla returned to pre-Blob levels within a couple of years after the heatwave, changes in the species composition of the sessile invertebrate community attributed to the Blob have persisted (PERMANOVA, F = 13.462, p < 0.001; Fig. 4 ). As positive temperature anomalies subsided in early 2016, invertebrate cover and richness steadily increased over time, peaking in the summers of 2017 and 2018 (Fig. 1 ). This increase in invertebrate cover was largely driven by increases in the abundance of bryozoans, and to a lesser extent, mollusks (Fig. 3 , Supplementary Fig. 2 , Supplementary Fig. 3 , Supplementary Fig. 4 ). Two invasive bryozoan species accounted for much of this increase (Fig. 4 ). The percent cover of Watersipora subatra , a recent invader in the Santa Barbara Channel 30 , increased after the Blob (Supplementary Fig. 3 , Fig. 4 ; IndVal = 0.455, p < 0.001), while Bugula neritina , a long-established invader, is now substantially more abundant than its morphologically similar native relative, Bugulina californica (Supplementary Fig. 3 , Fig. 4 ; IndVal = 0.549, p < 0.01). The increased abundance of W. subatra and B. neritina following the Blob could be due to reductions of specialized native species that otherwise would outcompete generalist invaders 31 , increased tolerance to thermally or metabolically stressful conditions, as has been demonstrated for W. subtorquata and some invasive ascidian species 32 , or increased recruitment of these species 33 . Fig. 4 Canonical Analysis of Principal Coordinates (CAP) of species composition of kelp forest sessile invertebrate communities. a CAP with time relative to the Blob (before: 2008–2013, during: 2014–2015, and after: 2016–2021) as a constraining variable. Points represent the average species composition across four sites for each sampling season. Lines represent ( b ) prominent species with correlations >0.6; (1) Actinostella californica (Phylum Cnidaria) (2) Bugula neritina (Phylum Bryozoa) (3) Thylacodes squamigerus (Phylum Mollusca) (4) Watersipora subatra (Phylum Bryozoa) (5) Diaperoforma californica (Phylum Bryozoa) (6) Phragmatopoma californica (Phylum Annelida) (7) Timarete luxuriosa (Phylum Annelida) (8) Encrusting bryozoan spp. (Phylum Bryozoa). In addition to increases in nonnative species, the abundance of a native southern-affinity sessile gastropod, Thylacodes squamigerus , increased significantly since the onset of the Blob (IndVal = 0.711, p < 0.001; Fig. 4 , Supplementary Fig. 4 ). As one of the few locally abundant invertebrates with a southern range extending beyond Baja California, T. squamigerus may have been preadapted to warmer temperatures. T. squamigerus also has the capacity to consume kelp detritus as an alternative food source 34 , which may have facilitated its survival during extended periods of low plankton availability. Though most sessile invertebrates are suspension feeders relying on the delivery of plankton and particulate organic matter for food 10 , 11 , there are differences in life history traits and feeding strategies among benthic phyla that may result in unequal responses to marine heatwaves. Colonial species, including sponges, most bryozoans, and many ascidians, are generally shorter-lived and exhibit rapid growth rates. By contrast, anthozoan cnidarians and mollusks are generally longer-lived with slower growth 35 , and may have specific traits (e.g., lower metabolism, energy stores, alternate feeding strategies) that enable them to survive prolonged periods of warming with anomalously low phytoplankton supply. For example, anemones are opportunistic feeders that can consume zooplankton and detritus 36 , while colonial species such as bryozoans and ascidians may be more dependent on smaller phytoplankton 37 , with a lower capacity to switch food sources in the event of low phytoplankton abundance. Though adult anthozoans and mollusks were generally resilient during the Blob, prolonged reductions in phytoplankton could adversely affect their populations by limiting recruitment in species with planktotrophic larvae that depend on phytoplankton for food. Increases in bryozoan cover following the Blob, particularly in two invasive species, suggests that rapid reproduction and growth may have facilitated their colonization following disturbance and increases in unoccupied space. Similar trends were observed in Mediterranean temperate reefs where traits associated with rapid growth and reproduction were significantly more common in benthic assemblages following heatwaves 38 . Suspension-feeding invertebrates are an essential link between pelagic and benthic food webs 10 , are important fisheries and aquaculture species, and are especially susceptible to environmental stressors due to the constraints in food acquisition and reproduction imposed by sessility. We found large negative impacts of warming and accompanying phytoplankton decreases on benthic suspension feeders along with persistent changes in their species composition in kelp forests of southern California, even though the major habitat-forming species, giant kelp, was relatively resilient to warming 18 , 26 . The Blob had similar effects in altering the species composition (but not community biomass) of understory macroalgae at our study sites 18 , 39 , which in turn, may have indirectly affected the sessile invertebrate community. We predict that increasing marine heatwaves will result in future losses and changes in species assemblages of sessile animals on temperate rocky reefs worldwide, and these transformations will likely be exacerbated by warming-induced declines in structure-forming kelps and understory macroalgae. Declines or changes in the species composition of suspension feeders may disrupt coastal food webs if the “winners” are functionally dissimilar from existing taxa. For example, heatwaves have altered functional traits in temperate reef systems through changes to benthic community structure 38 . Additionally, introductions of tropical herbivorous fish to temperate reefs during reef tropicalization have resulted in herbivore-mediated declines of macroalgae 40 , altering ecosystem function. Such functional shifts may have significant consequences for temperate reefs, including decoupling of primary production from higher trophic levels that depend on these vital primary consumers for survival."
} | 4,418 |
40251215 | PMC12008304 | pmc | 6,095 | {
"abstract": "Microbial community responses to environmental stressors are often characterised by assessing changes in taxonomic structure, but such changes, or lack thereof, may not reflect functional changes that are critical to ecosystem processes. We investigated the individual and combined effects of nutrient enrichment ( + 10 mg/L N, + 1 mg/L P) and salinisation ( + 15 g/L NaCl)—key stressors in freshwater systems—on the taxonomic structure and metabolic function of benthic microbial communities using 1000 L open freshwater ponds established >10 years ago in the field. Combined stressors drove strong decreases in maximum and mean total carbon metabolic rates and shifted carbon metabolic profiles compared to either stressor individually and compared to ambient conditions. These metabolic functional changes did not recover through time and occurred without significant alterations in bacterial community taxonomic structure. These results imply that critical functions, including organic carbon release, are likely to be impaired under multiple stressors, even when taxonomic structure remains stable.",
"introduction": "Introduction Community structure and function are central properties of natural systems used to understand and quantify ecosystem change under environmental stress 1 – 3 . Microbial communities underpin key ecosystem functions (e.g. nutrient and carbon cycling, primary productivity); thus, changes in microbial community structure and function can be indicators of ecosystem responses to environmental stress 4 – 7 . Changes in the taxonomic structure of microbial communities, which we define as the composition and abundances of taxa, can directly impact microbial functions, which we define as the sum of the activities performed by all members of the community, which in turn can lead to broader ecosystem-level changes 8 – 10 . For example, shifts in bulk soil microbial community structure under climate change-related stressors can result in changes to decomposition rates of organic carbon in soils 11 , 12 . However, stressors can also affect community structure and function independently of each other. Functional changes may occur without corresponding shifts in community structure when stressors induce sub-lethal physiological effects on individual organisms 13 , for example, where stressors induce dormancy in a portion of the community 14 . Conversely, structural changes of microbial communities might not translate into functional changes if, for example, the new/modified microbial community can still perform the same functions, i.e. the system exhibits functional redundancy 15 , 16 . Both scenarios can be common in microbial communities due to their high taxonomic diversity, physiological plasticity and rapid reproduction rates 15 – 18 . Furthermore, disturbances can affect the structure and function of microbial communities in different ways, resulting in different trajectories over time 8 , 19 – 21 . For example, in aquatic systems, bacterial community structure tends to continue to drift away from the pre-disturbance state after a disturbance 22 , while community function often recovers through time 23 . Despite this complexity, predictions about the functional consequences of environmental stressors frequently rely on assessments of microbial community structure alone which may not translate into changes in microbial function, limiting our ability to accurately predict and mitigate the effects of environmental stressors 21 , 24 . Freshwater lakes and ponds are important habitats that support biodiversity, primary productivity, carbon cycling and water supply 25 , 26 . Climate and land-use changes are causing multiple environmental stressors to interact, leading to novel and unpredictable ecological responses in the community structure and function of freshwater ecosystems 19 , 27 – 30 . Multiple freshwater stressors interact to affect community composition across various levels of biological organisation, for example macroinvertebrates 31 , 32 , diatoms 29 , periphyton 30 , bacterioplankton 33 , bacteria 34 and fungi 35 , and drive non-additive effects on important functions, for example, organic matter decomposition 27 , 28 , organic substrate utilization 36 and phosphorous uptake 32 . While stressor interactions have been studied extensively in freshwater systems, combined effects of stressor pairings involving salinisation, an emerging freshwater threat, remain understudied (for notable exceptions, see 32 , 37 ) and have been highlighted as a significant research priority 38 . In addition, most studies testing the effects of freshwater stressors apply treatments to developing communities or newly developed communities (i.e. <1 yr of succession), limiting our understanding of how stressors affect well established communities reflective of realistic field scenarios. Freshwater salinisation is increasingly occurring across a diverse range of environmental contexts due to climate change, mining, agricultural runoff, freshwater extraction, drought and other human activities, and is threatening freshwater ecosystems globally 38 . Salinisation and nutrient enrichment often occur in combination 32 , 37 , yet their combined effects on freshwater microbial communities are not well understood. This is important because changes to microbial community structure and function may translate into changes in ecosystem function 6 , and understanding the changes in microbial community structure and function under these stressors individually and in combination may inform management, for example, in the design of meaningful microbial bioindicators 39 . Increased salinity causes osmotic stress which typically reduces microbial growth, biofilm formation and overall microbial abundance 24 , 40 , 41 , alters community structure in favour of euryhaline organisms 40 , 42 , 43 and decreases rates of key microbially mediated functions, such as organic carbon decomposition 44 – 46 . In contrast, increased nutrient concentrations typically increase microbial biomass, bacterial growth rates and biofilm thickness 47 – 49 , shift community structure in favour of fast growing copiotrophic bacterial taxa resulting in homogenisation 50 – 52 and increase rates of key microbial functions such as production of carbon degrading extracellular enzymes, organic carbon decomposition and community respiration 49 , 53 , 54 . Given the effects of these stressors on microbial communities are mediated by different mechanisms, their interactive effects may result in complex structural and/or functional responses which are hard to predict by studying each of these stressors in isolation. This is particularly important in the context of freshwater quality monitoring where there is increasing interest in the utility of microbial bioindicators under multiple stressor scenarios 39 . Although molecular advances have meant microbial community composition data can be readily obtained using culture-independent methods, approaches that quantify functional groups, genes or direct functional measures may be more meaningful indicators of environmental stress than broader community indices (e.g. richness or diversity) 39 . Generally, the magnitude of functional changes in an ecosystem is predicted to increase with the number of environmental stressors experienced 55 , 56 . The effects of multiple environmental stressors can be additive (i.e. the sum of the individual stressor effects) 54 or can result in more complex, non-additive interactions 19 , 57 – 60 . These non-additive interactions can be positively or negatively synergistic (where the combined effect is more positive, or more negative, than predicted additively, respectively), or positively or negatively antagonistic (where the combined effect is less positive, or less negative, than predicted additively) 61 . Multiple stressor effects can change through time 36 and differ depending on the context and the functions being quantified. For example, inorganic nutrient enrichment and increased temperature may have synergistic effects on microbial organic carbon decomposition rates in soils (i.e. the magnitude of change is greater than the sum of the individual stressor effects) 62 , but antagonistic effects on microbial productivity in aquatic systems (i.e. the magnitude of change is less than the sum of the individual stressor effects) 63 . Critically, most studies investigating microbial community responses to multiple stressors have been conducted using simplified communities and in controlled environmental conditions (but for an example of a multiple stressor experiment in complex freshwater communities, see 27 ). Thus, to better predict how nutrient enrichment and salinisation impact the structure and function of complex microbial communities in realistic settings, further research is needed under realistic and ecologically relevant environmental change scenarios with established communities (i.e. >1 year of succession). This study assessed the independent and interactive effects of two relevant stressors in freshwater systems—salinisation and nutrient enrichment—on benthic microbial communities in semi-natural freshwater ponds (open ponds with established communities in the field for >10 years) 64 , 65 . Specifically, we determined whether microbial community functional changes in response to environmental stressors occurred in conjunction with, or independently from, community structural changes and whether this was consistent among stressor types, stressor combination and at multiple time points. We manipulated concentrations of salts and inorganic nutrients in the ponds, both individually and in combination. Microbial community structure and bacterial abundance were quantified via amplicon sequencing and qPCR of the 16S rRNA gene and measurements of the concentration of phototrophic groups (green algae, diatoms and cyanobacteria) using pigment fluorescence. Community-level function was quantified using community level physiological profiling of metabolic rates on a diverse array of carbon sources and via measurements of community respiration rate, net and gross primary productivity, biofilm biomass and photosynthetic efficiency, providing a comprehensive representation of key microbial functions in freshwater systems 66 – 68 . Elevated salinity and nutrient enrichment can both have strong effects on freshwater microbial community taxonomic structure, and environmental stress-induced changes to taxonomic structure generally strengthen through time. In contrast, these stressors generally have opposing functional consequences (elevated salinity tends to decrease overall microbial community activity, while nutrient enrichment tends to increase activity) which often recover through time. Therefore we hypothesised that both increased salinity and nutrients would change microbial community structure compared to ambient conditions, and that the magnitude of these structural shifts would increase through time, indicating community drift, and be greatest where both stressors were combined. Given the differences in mechanisms by which each of these stressors generally affect microbial communities, we also predicted that increased salinity would decrease rates of microbial function, including the rates of carbon metabolism, community respiration, community net and gross primary productivity, biofilm biomass and photosynthetic efficiency, while increased nutrients would increase these microbial functional metrics, resulting in a smaller effect on microbial function where both stressors were combined. Finally, we predicted that the magnitude of functional changes would decrease through time, indicating recovery.",
"discussion": "Discussion We found a strong combined effect of elevated salinity and nutrient enrichment on the metabolic function of freshwater benthic bacterial communities. Community level physiological profiles differed, and total mean and maximum carbon metabolic rates were lower in the combined stressor treatment compared to either stressor in isolation and compared to ambient conditions, with carbohydrate, carboxylic acid and amino acid metabolism most affected. Contrary to our predictions, the magnitude of these stress-induced changes in metabolic function did not recover through time. Together these results show that the effects of multiple stressors on bacterial community metabolism were stronger than individual stressor effects and could not be predicted from examining effects of each stressor in isolation. Contrary to our predictions we did not see strong effects of stressors on bacterial community taxonomic structure, although there was an initial trend for decreased bacterial diversity under the combined stressor treatment, and some effects of treatment on the temporal changes in concentration of green algae throughout the experiment. This suggests the metabolic functional responses of freshwater bacterial communities may be more sensitive to environmental stressors than bacterial community taxonomic structure and other broad compositional metrics (e.g. concentration of groups of phototrophic organisms), emphasising the importance of direct functional measures. The magnitude of metabolic functional changes in benthic bacterial communities was greatest under combined nutrient enriched and elevated salinity conditions. Bacterial community metabolic rates increased with increased nutrient concentrations, although this was only evident after 30 days. This supports the general finding that nutrient enrichment can enhance bacterial community metabolic functional rates by overcoming resource limitations on microbial activity 49 , 53 , 54 , but contrasts with the finding that nutrient enrichment causes rapid, short-lived effects on aquatic bacterial community functions which typically recover over time 48 . This metabolic effect coincided with green algae concentrations also being significantly higher in the nutrient enriched treatment compared to elevated salinity and combined stressors, suggesting broad measures of taxonomic structure (i.e. concentration of groups of phototrophic organisms) may correlate with metabolic function under some stressor scenarios. There was a trend of decreased metabolic rates under elevated salinity, which may have been due to intracellular osmotic stress, although this effect was not statistically significant. Metabolic rates decreased faster (after one day) and to a greater magnitude under the combined stressor treatment compared to under either stressor individually. This supports the growing evidence that multiple stressors often have strong combined effects in aquatic ecosystems 19 , 57 – 60 and extends this trend to the metabolic function of freshwater benthic microbial communities. Under combined stressors, community level physiological profiles also shifted compared to all other treatments. This suggests that when the community was exposed to the two stressors with contrasting mechanisms of interference, the cumulative stress induced a major shift in the relative use of different carbon sources which did not occur under each stressor individually. This physiological shift in metabolic profile provides a potential mechanistic underpinning for the synergistic stressor interaction. Changes in metabolic rates under the stressor treatments were carbon group specific. Under conditions of combined elevated salinity and nutrient enrichment, rates of metabolism for carbohydrates, carboxylic acids and amino acids decreased significantly, while no significant differences were observed for polymers, phenols or amines. This pattern aligns with established findings that carbohydrate, carboxylic acid and amino acid metabolism in aquatic biofilm communities tend to be more sensitive to environmental stressors compared to the metabolism of polymers, phenols and amines 69 , 70 . One explanation for this heightened sensitivity lies in the composition of extracellular polymeric substances (EPS) within the biofilm matrix, which is predominantly made up of polysaccharides, proteins and carboxylic acids 71 . Consequently, biofilms are typically rich in extracellular enzymes that target these EPS compounds, including carbohydrates, amino acids and carboxylic acids 71 . Under conditions of combined elevated salinity and nutrient enrichment, the production of these extracellular enzymes may have diminished as microbial energy resources were redirected towards mechanisms of stress tolerance, potentially driving the observed significant decreases in the metabolism of these carbon groups. Rates of microbial carbon metabolism and the carbon use profiles of microbial communities can be key determinants of decomposition rates and total organic carbon release for higher organisms. While these processes are well studied in terrestrial soils 72 , 73 , they are increasingly recognised in freshwater systems 74 , 75 . These results imply that under multiple stressor scenarios carbon metabolism profiles will shift and overall rates of organic carbon metabolism, including organic matter decomposition and organic carbon release, are likely to decrease. However, while our study provides insights into the initial and intermediate responses of bacterial community metabolic functions, these functions were not measured at the 90-day time point, which restricts our ability to fully assess long-term functional dynamics. Future studies would benefit from quantifying the carbon metabolic profiles and rates of benthic bacterial communities across longer time periods to capture comprehensive temporal trends. The community level metabolic changes observed under nutrient enrichment and salinisation were not associated with changes in overall community productivity as quantified by total biofilm biomass, photosynthetic efficiency, community respiration, net primary productivity and gross primary productivity. This suggests the stressors interfered with the metabolic physiology of the community without impairing overall productivity. This stability in productivity may be explained by niche partitioning, a compensatory mechanism where the decreased productivity of sensitive organisms is balanced by the increased productivity of resistant organisms 76 – 79 . For example, the decreased metabolism of organic carbons observed under salinisation may have been balanced by an increase in the activity of autotrophs and/or organisms with a non-organic carbon metabolic preference resulting in no net change to overall community productivity. However, total biofilm biomass, as measured by chlorophyll a concentration, decreased throughout the experimental period and was significantly lower on day 90 compared to all other time points for all treatments. Similarly, the efficiency of community respiration also significantly decreased through time. This decrease in total biofilm biomass coincided with a significant decrease in the oxygen concentration in pond water through time, which turned largely anoxic by day 90. Therefore, it is possible that environmental changes, such as oxygen depletion, that impacted all treatment ponds equally may have masked potential treatment effects on biofilm functional metrics such as biomass and respiration rates. Contrary to our expectations, stressors had no impact on community structural metrics, including bacterial ASV abundance, diversity, structure and composition, although a trend was observed for decreased bacterial diversity under the combined stressor treatment. Several possible explanations could account for the unexpected resistance (i.e. no observed change) of bacterial community structure to all stressor regimes. First, once established, biofilms can exhibit strong structural stability and resistance to environmental disturbances 80 . The 30-day development period in ambient conditions, prior to the application of treatments, may have allowed biofilms to develop a highly resistant protective extracellular matrix enabling structural stability as has been observed for biofilms under various extreme stressor scenarios 81 . The procedure of gently shaking the tiles to remove sediment and loosely attached organisms may have had some disturbance on organisms attached to the outer layer of the biofilm structure. Bacteria that had adhered to the outer layers of the biofilm and were in direct or close contact with the water-column stressors may have shown the strongest taxonomic changes; however, these loosely attached microorganisms would have been more likely representative of assemblages in the water column, rather than the benthic microbial community, which was the target in our study and for which we did observe functional effects of treatments. Second, dormancy, a common microbial response to environmental stressors, might have resulted in treatment effects being masked by the retention of DNA from dormant or dead cells in the extracellular biofilm matrix 14 , 82 . Finally, the four independent ambient ponds in which biofilms developed were established ten years ago, which may have resulted in different ecological trajectories that may have introduced variation in community assembly and bacterial community structure on the tiles at the start of the experiment. These differences in initial community assembly may have introduced variability in community responses to stressors, and this variability may have decreased statistical power to detect treatment effects (if any). Regardless of the cause of the lack of treatment effects observed in the bacterial taxonomic structure, our results indicate that bacterial community structure based on DNA amplicon sequencing is not always a strong indicator of freshwater community metabolic responses to environmental stressors. These findings challenge the notion that DNA-based measures of bacterial community taxonomic structure are key indicators of functional change and instead supports literature suggesting that microbial structure and functional responses to stressors often occur independently 19 , 57 – 60 . This study demonstrates that the effects of two key global freshwater stressors, nutrient enrichment and salinisation, on benthic bacterial community metabolic function were stronger where stressors were combined compared to individual effects, and that these functional effects occurred largely independently of changes in bacterial community taxonomic structure. These results suggest that as freshwater stressors increasingly co-occur under global change scenarios, the metabolic profiles of benthic bacterial communities will likely shift and overall rates of organic carbon metabolism will decrease, potentially affecting organic matter decomposition, organic carbon release and the freshwater carbon cycle. In addition, these results suggest that future research on freshwater benthic microbial communities and other similarly resistant communities (e.g. environmental biofilms) should prioritise direct measurements of microbial community functions when assessing microbial responses to environmental stressors, rather than inferring functional change solely from shifts in bacterial community taxonomic structure."
} | 5,795 |
39005283 | PMC11244974 | pmc | 6,097 | {
"abstract": "Microbial communities vary across space, time, and individual hosts, presenting new challenges for the development of statistics measuring the variability of community composition. To understand differences across microbiome samples from different host individuals, sampling times, spatial locations, or experimental replicates, we present FAVA, a new normalized measure for characterizing compositional variability across multiple microbiome samples. FAVA quantifies variability across many samples of taxonomic or functional relative abundances in a single index ranging between 0 and 1, equaling 0 when all samples are identical and equaling 1 when each sample is entirely comprised of a single taxon. Its definition relies on the population-genetic statistic F S T , with samples playing the role of “populations” and taxa playing the role of “alleles.” Its convenient mathematical properties allow users to compare disparate data sets. For example, FAVA values are commensurable across different numbers of taxonomic categories and different numbers of samples considered. We introduce extensions that incorporate phylogenetic similarity among taxa and spatial or temporal distances between samples. We illustrate how FAVA can be used to describe across-individual taxonomic variability in ruminant microbiomes at different regions along the gastrointestinal tract. In a second example, a longitudinal analysis of gut microbiomes of healthy human adults taking an antibiotic, we use FAVA to quantify the increase in temporal variability of microbiomes following the antibiotic course and to measure the duration of the antibiotic’s influence on microbial variability. We have implemented this tool in an R package, FAVA, which can fit easily into existing pipelines for the analysis of microbial relative abundances.",
"introduction": "Introduction Understanding the compositional variability of microbial communities across space, time, or host individuals is important for characterizing these communities and their relationships with biological variables of interest ( Turnbaugh et al., 2007 ; Dethlefsen and Relman, 2011 ; Faith et al., 2013 ; David et al., 2014 ; Flores et al., 2014 ; Coyte et al., 2015 ; Oh et al., 2016 ; Thompson et al., 2017 ; Goldford et al., 2018 ; Ji et al., 2019 ; Fassarella et al., 2021 ; Estrela et al., 2022 ; Upadhyay et al., 2023 ). For example, studies of microbiome composition have found that microbiome compositions are often more variable across dysbiotic individuals than across healthy individuals ( Zaneveld et al., 2017 ), the microbial communities of infants tend to be more variable across individuals than those of adults ( Kurokawa et al., 2007 ), and gut and tongue microbiomes that are more diverse may be less temporally variable ( Flores et al., 2014 ). Despite its biological importance, however, compositional variability is difficult to directly quantify with existing methods. We define “compositional variability” as variability across two or more compositional vectors—lists of proportions that sum to 1 ( Figure 1A ). Compositional variability is minimized when the compositional vectors have identical compositions; it is maximized when each vector contains a single category at 100% frequency ( Figure 1B and C ). We focus on vectors that represent the composition of microbiome samples. These vectors’ entries represent relative abundances of taxonomic categories such as OTUs, species, or even functional categories such as gene classifications, inferred from 16S or metagenomic sequencing data ( Lozupone et al., 2012 ; Louca et al., 2017 ; Shalon et al., 2023 ). Each vector can represent the composition of a microbiome sample from a distinct timepoint, spatial location, host individual, or replicate. Compositional variability can therefore represent temporal stability, spatial heterogeneity, inter-host diversity, or repeatability ( Faith et al., 2013 ; Bashan et al., 2016 ; Goldford et al., 2018 ; Mehta et al., 2018 ; Seekatz et al., 2019 ; Sheth et al., 2019 ; Roodgar et al., 2021 ; Estrela et al., 2022 ; Guthrie et al., 2022 ; Shalon et al., 2023 ) Traditionally, microbiome studies have used statistics such as the Shannon and Gini-Simpson indices ( Patil and Taillie, 1982 ), the Jensen-Shannon divergence ( Lin, 1991 ), and the Bray-Curtis dissimilarity ( Bray and Curtis, 1957 ). Single-sample diversity statistics such as the Shannon and Gini-Simpson indices quantify the variability of microbiome samples considered individually, answering questions such as “Which of these microbiomes is the most diverse?” Pairwise statistics, such as the Jensen-Shannon divergence, Jaccard index, and Bray-Curtis dissimilarity compare the compositions of two samples, answering questions such as “How does the composition of a perturbed microbial community compare to a pre-perturbation reference sample?” Although these tools are valuable when variability is of interest in one sample or between two samples, they are less well suited to scenarios in which three or more samples are of interest, as they only consider one or two samples at once. Studies that seek to quantify variability across many samples are often limited to computing summary measures of each sample, such as diversity indices ( Flores et al., 2014 ; Shalon et al., 2023 ), principal component coefficients ( Zaneveld et al., 2017 ; Olm et al., 2022 ), or the abundances of individual taxa ( Ji et al., 2019 ; Kang et al., 2022 ), and computing the variability across samples of these summary statistics. However, this approach measures the variability of a summary statistic, not the variability of the microbiome composition itself. Because it is possible for very different compositions to produce similar values of a summary statistic, such indirect variability measures potentially obscure large differences among samples. Existing methods are therefore insufficient to, for example, determine if a microbiome is more temporally variable after than before a perturbation, or to identify which of many anatomical regions has the most across-individual microbiome variability. These questions require statistics of compositional variability that are suited to arbitrary numbers of samples. Consider for illustration the study of Flores et al. ( Flores et al., 2014 ), which aimed to compare regions of the body in terms of their temporal variability in microbiome composition. For 85 adults, they profiled the microbiomes of four body habitats weekly for three months. They measured temporal variability by computing diversity statistics such as the Shannon index for each temporal sample, then computing the coefficient of variation of the Shannon index over time for each of the 85 individuals and four body regions. This approach quantifies the variability of the Shannon diversity, not the variability of the microbiome composition itself. Because microbiomes with either no species or many species in common could have identical Shannon indices, this method could assign time series with dramatically different compositional change the same coefficient of variation, obscuring meaningful differences. In this paper, we present FAVA, a statistical measure that quantifies variability of microbiome composition across many microbiome samples. In a single number, FAVA measures variability of microbial composition across arbitrarily many microbiome samples, providing a summary of large data sets. The measure allows for the optional inclusion of similarities among taxonomic categories (e.g., phylogenetic similarity) as well as for optional non-uniform weighting of samples (e.g., to account for uneven sampling time intervals). FAVA, which stands for an F ST -based A ssessment of V ariability across vectors of relative A bundances, is based on the population-genetic statistic F S T , which is traditionally used to quantify variability across vectors of allele frequencies for multiple populations. FAVA takes values between 0 and 1, equaling 0 when all sampled microbiome compositions are identical, and equaling 1 when each sample contains only a single taxon and at least two distinct taxa are present across samples ( Figure 1B and C ). Because FAVA is a normalized statistic, it can be used to compare variability among sets of samples with very different numbers of taxa or datasets with very different numbers of samples. We demonstrate FAVA with two datasets, one containing spatial samples along the gastrointestinal tract of seven species of ruminants, and the other describing time series of microbiome samples from 22 human individuals who experienced an antibiotic perturbation. In the ruminant dataset, we identify substantially higher inter-individual variability in the stomach and small intestine than in the large intestine, supporting the view that substantial microbiome variability is obscured when gastrointestinal communities are sampled through fecal samples alone ( Shalon et al., 2023 ). In the human dataset, we show that temporal variability in microbiome composition is significantly elevated following an antibiotic perturbation, and that just half of subjects return to low levels of temporal variability in the 30 days following completion of the antibiotic.",
"discussion": "Discussion We have introduced an index to quantify variability across many samples of microbiome composition. We defined the measure through an analogy with the population-genetic statistic F S T , considering microbiome samples in place of populations and microbial taxa in place of alleles. FAVA, an F S T -based Assessment of Variability across vectors of relative Abundances, equals 0 if and only if all microbiome samples are identical, and equals 1 if and only if each microbiome sample contains only a single taxon and there is more than one taxon present across all samples ( Figure 1 ). FAVA can be used as a measure of compositional variability across time points, spatial sampling locations, host individuals, or replicates, quantifying the temporal variability, spatial heterogeneity, or replicability of microbial communities. Because FAVA takes values between 0 and 1 regardless of the number of sampled taxa, we can compare values of FAVA between very different data sets, such as data on abundances of different taxonomic categories. To demonstrate FAVA’s performance as a measure of microbiome variability across many samples, we analyzed two microbiome data sets: an investigation of ruminant microbiome composition along the gastrointestinal tract ( Xie et al., 2021 ), and a longitudinal study of human gut microbiome composition before and after an antibiotic perturbation ( Xue et al., 2023 ). In the ruminant data, we found that compositional variability across individuals—either within a host species or across host species—was consistently lower at the end of the gastrointestinal tract than in the middle, supporting the view that substantial inter-individual heterogeneity is missed when microbiomes are monitored by fecal sampling alone ( Figure 2B and D ) ( Shalon et al., 2023 ; Tropini et al., 2017 ). We found that, in all gastrointestinal regions, taxonomic abundances were much more variable across individuals than were functional abundances, a result that corroborates observations of microbial functional redundancy in the gastrointestinal tract ( Figure 2D ) ( Louca et al., 2018 ). In the human microbiome data, we found that antibiotic perturbations significantly destabilize microbial communities, resulting in elevated temporal variability following an antibiotic ( Figure 4E ). Computing FAVA in sliding windows across temporal samples for each subject increased the granularity of this analysis. We found that, although elevated variability lasted for only 1-2 weeks post-antibiotic on average, few subjects returned to their pre-antibiotic variability levels during the study duration ( Figure 4C and D ). We also highlighted FAVA’s ability to quantify temporal variability separate from compositional state by focusing on subjects XDA and XMA, who returned to their pre-antibiotic variability levels ( Figure 4C ) even though only subject XMA returned to the original composition ( Figure S3 ). We introduced two extensions of FAVA: weighted FAVA ( equation 11 ), which can incorporate both similarity among taxa and distance between samples into the computation, and normalized FAVA, which accounts for the abundance of the most abundant taxon, allowing for more meaningful measurement of variability across small numbers of samples. In our analysis of human gut microbiome data over time ( Xue et al., 2023 ), the use of weighted FAVA helped to account for both the combination of weekly and daily samples and the broad range of species appearing in the data. FAVA values can be influenced by the choice of weights. For example, Figure S4 presents two simple hypothetical OTU tables with a difference in FAVA of nearly 0.5 when weighted by taxonomic similarity, despite having identical unweighted FAVA values. Nevertheless, in our analysis of human microbiome data, although individual FAVA values shift with the incorporation of weights, FAVA values computed across post-antibiotic samples are consistently higher than those computed across pre-antibiotic samples, regardless of whether FAVA is weighted by sampling times, taxonomic similarity, or both ( Figure S5 ). Our measure, which we have implemented in an R package, contributes to a large body of methods for the analysis of microbiome relative abundance data ( McMurdie and Holmes, 2013 ; Bolyen et al., 2019 ). We emphasize, however, that FAVA is a multi-sample compositional variability measure, setting it apart from the many existing measures of pairwise compositional similarity, such as Unifrac, Bray-Curtis dissimilarity, and the Jensen-Shannon divergence ( Figure 1A ) ( Bray and Curtis, 1957 ; Lin, 1991 ; Lozupone and Knight, 2005 ). For example, two separate collections of microbiome samples can have identical values of FAVA, but wildly different mean compositions (e.g., Figure 4B and C ). Similar values of FAVA therefore reflect similarities in the spatial or temporal dynamics shaping variability, not compositional similarity. FAVA builds on a rich literature of population-genetic and ecological frameworks for hierarchical partitioning of genetic, taxonomic, and phylogenetic diversity across individuals and communities ( Lewontin, 1972 ; Lande, 1996 ; Ricotta, 2005 ; Hardy and Senterre, 2007 ; Ellison, 2010 ). Indeed, F S T has sometimes been used as a measure of compositional variability in ecological contexts ( Gilbert and Levine, 2017 ). Future applications of FAVA can span the range of questions that researchers pose about compositional variability, from understanding temporal variability in infant microbiomes ( Koenig et al., 2011 ; Yassour et al., 2016 ) to quantifying the repeatability of community assembly across experimental replicates to identifying the timing of compositional stability in serial passaging experiments ( Goldford et al., 2018 ; Estrela et al., 2022 ). Because FAVA measures a fundamentally different phenomenon relative to existing methods for microbiome analysis, it opens up a suite of novel research questions relating to temporal stability, individual heterogeneity, spatial variability, and replicability."
} | 3,841 |
24408876 | PMC4060947 | pmc | 6,100 | {
"abstract": "Synechocystis sp. PCC 6803 is the most popular cyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insights into their metabolic potentials and regulatory networks. To enable a comparative approach, we generated the draft genome sequence of Synechocystis sp. PCC 6714, a closely related strain of 6803 (16S rDNA identity 99.4%) that also is amenable to genetic manipulation. Both strains share 2838 protein-coding genes, leaving 845 unique genes in Synechocystis sp. PCC 6803 and 895 genes in Synechocystis sp. PCC 6714. The genetic differences include a prophage in the genome of strain 6714, a different composition of the pool of transposable elements, and a ∼40 kb genomic island encoding several glycosyltransferases and transport proteins. We verified several physiological differences that were predicted on the basis of the respective genome sequence. Strain 6714 exhibited a lower tolerance to Zn 2+ ions, associated with the lack of a corresponding export system and a lowered potential of salt acclimation due to the absence of a transport system for the re-uptake of the compatible solute glucosylglycerol. These new data will support the detailed comparative analyses of this important cyanobacterial group than has been possible thus far. Genome information for Synechocystis sp. PCC 6714 has been deposited in Genbank (accession no AMZV01000000).",
"introduction": "1. Introduction Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insight into the metabolic potentials and regulatory networks. Among cyanobacteria, this has been illustrated by detailed comparative analyses of the marine picoplanktonic cyanobacteria Prochlorococcus and Synechococcus. 1 – 3 However, due to the lack of data from closely related strains, no comprehensive comparison has focused on Synechocystis sp. PCC 6803 (from here on Synechocystis 6803), the otherwise most popular cyanobacterial system to work with. Synechocystis 6803 was the first phototrophic and the third organism overall for which a complete genome sequence was determined. 4 The genome of Synechocystis 6803 was manually curated by the research community at CyanoBase ( http://genome.microbedb.jp/cyanobase/Synechocystis) . 5 Over the years, several substrains of 6803 evolved in different laboratories showing distinct physiological features (e.g. glucose tolerance), from which also several have recently been re-sequenced. 6 – 9 The coverage with analysed genome sequences for the cyanobacterial phylum has been greatly improved recently. Based on a diversity-driven selection of species for genome sequencing, 54 additional strains were analysed, 10 raising the number of publicly available cyanobacterial genome sequences to 126. With strain PCC 7509 also, one Synechocystis strain was sequenced. However, it is only very remotely related (90% 16S rRNA identity) to Synechocystis 6803 and belongs even to another clade (B1) than Synechocystis 6803 (B2) in the cyanobacterial tree. 10 Therefore, despite its naming as Synechocystis , the strain PCC 7509 is quite distant from Synechocystis 6803. In the current cyanobacterial tree, Synechocystis 6803 is sharing a clade with unicellular N 2 -fixing oceanic strains such as Cyanothece spp. 10 It has been reported that a 97–100% 16S rRNA identity is necessary for a productive genome comparison among strains. 1 – 3 Thus, Synechocystis 6803 lacked a closely related organism with a known genome sequence that appeared suitable for comparative analysis. To fill this gap, we selected Synechocystis sp. PCC 6714 (from here: Synechocystis 6714) as candidate. Synechocystis 6803 as well as strain 6714 are unicellular cyanobacteria that were isolated from the same freshwater pond in Oakland, California, by R. Kunisawa. These strains were initially part of the ‘Berkeley Culture Collection’, 11 which were later transferred into the ‘Pasteur Culture Collection’ of cyanobacteria. 12 The decision to choose Synechocystis 6714 was further supported by the high 16S rRNA identity (99.4%) among the two strains, thus well suited for comparative analyses. Their close genetic relation also was seen in an expression-based screen that revealed the presence of a highly transcribed CRISPR system in it, 13 similar to the one in Synechocystis 6803. 14 Moreover, the strain 6714 also represents an established laboratory strain, amenable to genetic manipulation. 15 , 16 Here, we focus on the draft genome analysis of Synechocystis 6714 in comparison to strain 6803. In a parallel study, we will provide the primary transcriptomes of both strains under 10 different conditions using strand-specific cDNA sequencing.",
"discussion": "3. Results and discussion 3.1. Draft genome of Synechocystis sp. PCC 6714 The genome of Synechocystis 6714 was sequenced using two libraries of different lengths by paired-end sequencing and assembled into five scaffolds ranging from 46 504 to 2 984 476 nt in length. The DNA is characterized by an average GC content of 47.37%, which is very close to the value of 47.4% reported for strain 6714 in 1971 based on CsCl density gradient equilibrium centrifugation. 11 The longest scaffold C2 likely represents the major part of the chromosome, since it closely resembles the chromosome of strain 6803. Table 1 summarizes the main features of the draft genome. Since the scaffolds C0 and C4 carry tRNA genes and the majority of their protein-coding genes have orthologues on the chromosome of Synechocystis 6803, they also are likely part of the chromosome. This assumption also is in line with their 3 and 5% higher GC content compared with the scaffolds C1 and C3. For comparison, the GC content of the Synechocystis 6803 chromosome is 47.72%, whereas it also is lower for three out of the four large plasmids (pSYSX, 42.72%; pSYSA, 44.48%, pSYSM, 42.95%). 25 By combining the scaffolds C0, C2, and C4, we estimated the size of the Synechocystis 6714 chromosome to be around 3.45 Mb, which is fairly similar to the 3.57 Mb of Synechocystis 6803.\n Table 1. Summary of the Synechocystis 6714 draft genome main features Scaffold Size (nt) Genes GC % tRNA genes C0 189 995 183 47.79 2 C1 181 306 201 42.41 0 C2 2 985 628 3034 47.72 35 C3 46 504 45 44.25 0 C4 287 195 270 47.13 4 Total 3 690 628 3733 47.37 41 Compared with the rather high similarity of the chromosome size and coding capacity, the similarities of plasmid sequences were rather low between the two strains. Among the seven plasmids 25 – 28 of Synechocystis 6803, we found no significant similarities toward its plasmids pSYSG, pCC5.2, pCA2.4, and pCB2.4 in strain 6714. In contrast, sequences resembling about one-third each of pSYSA, pSYSM, and pSYSX of Synechocystis 6803 were detected in strain 6714 (Fig. 1 ). Thus, our draft genome points at a different composition or lower coding capacity of extrachromosomal plasmids in strain 6714 compared with strain 6803.\n Figure 1. Genome coverage based on circular genome plots of the Synechocystis 6803 chromosome and its four large plasmids pSYSA, pSYSG, pSYSM ,and pSYSX. Tracks from the outside show (1) regions with BLASTN hit in Synechocystis 6714 and identity between 50% (grey) and 100% (red); (2) CDS features from forward and reverse strand in Synechocystis 6803; (3) GC content. A marked difference between both strains exists in the number and types of mobile genetic elements (Table 2 ). Synechocystis 6803 possesses at least 134 genes encoding transposases. These transposases, which were identified by BLASTp searches against the ISfinder database, 21 requiring a BLASTp E -value of ≤10 –8 , were assigned to 11 different families, each containing 1–45 identical copies. The highest copy numbers were found for the IS630, IS5, and IS701 families of IS elements (Table 2 , Supplementary Table S2 ). In Synechocystis 6714, we identified only 32 transposase genes, which belong to only six different families. The highest copy numbers were found for the IS200/IS605 family and as before in strain 6803 for IS630 and IS5 families (Table 2 , Supplementary Table S3 ). At a first glance, this high divergence in the numbers and types of insertion sequences appears surprising, given the otherwise close relatedness among the two strains. However, this finding is in line with reports for the ISY203 group of elements (belonging to the IS4 family) that vary even among substrains of 6803. Four members of this IS element with identical nucleotide sequences were present only in the ‘Kazusa’ substrain, whereas they were absent in the genomes of other substrains. 29 \n Table 2. Types and numbers of IS elements found on basis of identified transposase genes IS element Strain 6803 Strain 6714 IS1 10 0 ISTcSa 3 0 IS3 1 0 ISL3 3 0 IS4 14 0 IS200/IS605 1 5 IS256 2 1 IS5 31 7 IS630 45 11 IS701 23 3 Tn3 0 2 ISLre2 1 0 Total 134 32 Using RBH, we identified 2838 orthologous protein-coding genes between Synechocystis 6714 and 6803, leaving 845 specific genes in strain 6803 and 895 specific genes in strain 6714. Thus, among the two strains, more than 75% of the genes are conserved. However, many of the strain-specific genes belong to gene families that were clustered as paralogues to pairs of orthologue genes when using the MCL algorithm, 20 indicating gene duplications, sequence, and probably also functional diversification. The full list of orthologue and paralogue genes between the two strains is presented in Supplementary Table S4 . In Supplementary Tables S5 and S6 , we present the lists of unique protein-coding genes. Only 537 of the 845 Synechocystis -6803-specific genes are located on the chromosome (=16% of all chromosomal protein-coding genes; Supplementary Table S5 ), whereas 308 of the genes lacking a clear orthologue (=76% of all plasmid-located protein-coding genes in 6803) can be explained by the strong differences in the plasmid-located gene pool. Moreover, it should be noted that the majority of strain-specific genes encodes for proteins of unknown function, i.e. the functional significance of the majority of differences is thus uncertain. 3.2. Large-scale differences between Synechocystis 6803 and Synechocystis 6714: unique genetic arrangements in a large genomic island and prophage Psy1 The higher number of transposon genes in Synechocystis 6803 is correlated with a low degree of syntheny between the two strains. Another situation exists with the rfb- gene cluster that differs entirely between the two strains and encodes several glycosyltransferases possibly involved in cell wall biosynthesis and the modification of cell surface properties. This region has features of a genomic island, since the adjacent genes are conserved between the two Synechocystis strains, but the GC content drops considerably (from 48 to 35%) within this region in both strains (Fig. 2 ). Genomic islands consist of sets of genes that become laterally transferred, belong to the flexible gene pool of a bacterial phylum and frequently provide a certain fitness advantage. 30 Accordingly, the most closely related homologues matching to these proteins are found in a wide variety of organisms. For the 50 genes located in the Synechocystis 6714 rfb gene cluster, the phylogenetically top-matching proteins belong to groups as diverse as Zetaproteobacteria, Bacilli, Clostridia, Armatimonadetes, Rhodopirellula, and Stigonematales cyanobacteria. The top-matching proteins against the Synechocystis 6803 rfb gene cluster proteins are of comparable diversity. A particular example is also the norf2 gene which was annotated on the basis of transcriptome data. 19 The most closely related proteins to Norf2 (Fig. 2 ) are annotated in Thiocapsa marina (69% identical and 86% similar residues) and several Thioalkalivibrio species, pointing further to the alien origin of this genomic region.\n Figure 2. A likely genomic island in two Synechocystis strains. A genomic segment of ∼40 kb from Synechocystis 6803 is shown with some genes annotated for orientation (EPS, exopolysaccharide export protein; CmcI, Cephalosporin hydroxylase protein; GT1, GT1 family of glycosyltransferases; GTA-GTB, fusion protein joining a glycosyltransferase family A with a glycosyltransferase family B domain; Norf2 is a 68 amino acid peptide-encoding gene originally predicted on basis of transcriptome data indicating the presence of an mRNA for this conserved reading frame). 19 Adjacent genes to this region are in the two strains of the gene pairs slr0976/slr0977 and sly1015510/sly1015500 encoding a DUF820 protein and an ABC transporter permease component; left side in 6803) and slr1084/slr1085 and sly1015040/sly1015030 (encoding a WcaF-type acyl transferase and a glycosyltransferase; right side in 6803). The GC % content, indicated by the green bars (each representing 1000 nt), drops considerably within this region. Thus, this region has features of a genomic island. The nucleotide identity to matching segments in the Synechocystis 6714 genome is colour coded (red >90%, light red >70%). The corresponding stretch in the Synechocystis 6714 genome encompasses genes sly1015490– sly1015020 , almost entirely belonging to the list of unique genes in that strain ( Supplementary Table S6 ). The proteins encoded by these genes are annotated as hypothetical proteins, UDP-glucose 4-epimerase, several different glycosyltransferases, rhamnogalacturonides degradation protein RhiN, dTDP-glucose 4′6′-dehydratase, methylase/methyltransferase, ABC transporter, GDP-mannose 4′6′dehydratase and as NAD-dependent epimerase/dehydratase. An example for genome scrambling worth mentioning exists in the hydrogenase operon that encompasses the seven genes sll1220–sll1226 ( hoxEFUYH plus two additional genes for proteins of unknown function) in strain 6803. In Synechocystis 6714, the orthologues of these seven genes ( sly1009900–sly1009960 ) form a cluster with gene sly1009870 encoding the NiFe hydrogenase metallocenter assembly protein HypD, whereas the homologue in Synechocystis 6803, slr1498 , is located 1.62 Mb away. A further difference between Synechocystis 6714 and 6803 genomes is the presence of a prophage in the former but its lack in the latter (Fig. 3 ). As this prophage has not been previously described, we called it Psy1, for prophage in Synechocystis 1. The genomic DNA of Psy1 has integrated into the trnF (phenylalanine-specific tRNA GAA ) gene, duplicating its 3′ half but restoring the gene to be functional intact. This insertion might have occurred only recently as the duplicated segment of the trnF gene is still sequence identical with the original prophage host gene. Although the Psy1 genome is with a total length of 20 660 nt quite short for a prophage, genomes of comparable size have recently been reported for siphoviruses, which infect marine cyanobacteria (e.g. S-CBS1 infecting Synechococcus strains CB0201, CB0204, CB0202, and CB0101). 31 The annotation of Psy1 adds another 27 genes unique for strain 6714 ( Supplementary Table S6 ). Most of these genes have no closely related homologues in database searches, indicating that Psy1 might belong to a novel group of bacteriophages. Clear homologues exist for Sly1027750, an integrase with several homologues in other cyanobacterial genomes; Sly1027640, an HK97 family phage portal protein with the tail sheath protein from the Pseudomonas transducing phage PhiPA3 as the best matching protein in the bacteriophage database (BlastP E -value 7e −16 ); 32 Sly1027490, a lysozyme superfamily protein with the putative endolysin from Acinetobacter phage phiAC-1 as the best matching bacteriophage protein (BlastP E -value 2e -30 ); 33 Sly1027700, a D5 N terminal like domain-containing protein of phage D5 proteins and bacteriophage P4 DNA primases (Fig. 3 ).\n Figure 3. Prophage Psy1 inserted into the trnF GAA gene (labelled by the green stars) of Synechocystis 6714. Genome position is drawn along the x -axis. Protein coding genes are shown in red if conserved in Synechocystis 6803 and in blue if not; trnF GAA is shown in green. Transcriptome read counts per 100 million for the forward strand are plotted above and for the reverse strand below the CDS features. The GC % content is indicated by the green bars (each representing 1000 nt). The following genes were annotated as coding for bacteriophage-related proteins and are mentioned in the text: sly1027640 , HK97 family phage portal protein; sly1027490 , bacteriophage lysozyme-like protein; sly1027670 and sly1027680, remotely similar to bacteriophage Cro repressor; sly1027700 , D5 N terminal like domain-containing protein of phage D5 proteins and bacteriophage P4 DNA primases; sly1027750 , phage integrase. Our complementary transcriptome data (unpublished) indicate that the Psy1 genes are not significantly expressed except for a short region, encompassing the two short genes sly1027670 and sly1027680 (Fig. 3 ). One of the two proteins encoded by these two genes, Sly1027680, has similarity to bacteriophage repressor proteins and belongs to the HTH XRE family of Cro/CI repressor proteins, suggesting its possible involvement in silencing Psy1 activity. The other protein, Sly1027670, possesses a predicted partial endoribonuclease Y domain and revealed in database searches several good matches, with protein Ssl7074 from Synechocystis 6803 as the top hit (47% identical and 59% similar positions). Interestingly, gene ssl7074 in Synechocystis 6803 is located within the CRISPR2-associated region of cas genes next to the cas 6-2b gene, a candidate for an endonuclease involved in CRISPR crRNA maturation. 14 3.3. Genetic differences with particular physiological relevance Several of the strain-specific genes likely affect the physiology or provide certain strain-specific characteristics allowing their settlement in specific environmental niches (see Supplementary Table S5 and S6 for the list of unique genes in Synechocystis 6803 and 6714, respectively). For instance, only Synechocystis 6803 possesses genes for the proteins Flv2 and Flv4, which are essential for growth under fluctuating light and are supposed to protect photosystem II against photoinhibition. 34 In contrast, in Synechocystis 6714, two operons are found, each encoding all subunits of the high-affinity K + transporter Kdp, 35 similar to the situation in filamentous cyanobacteria such as Anabaena sp. PCC7120, whereas Synechocystis 6803 harbours only one copy of the kdp2 type. 36 To date, it was believed that unicellular cyanobacteria have a single kdp system or none, whereas filamentous cyanobacteria have two or more copies. 36 A distinct group of protein-coding genes that differs between the two strains are associated with the CRISPR system, the prokaryotic immune system, accounting for 17 different proteins alone ( Supplementary Tables S5 and S6 ). There are three distinct loci of CRISPR- cas genes in both strains. 13 , 14 One of them (called CRISPR3/CRISPR3*) is highly conserved, whereas the other two appear to have been substituted over their entire length, possibly by an active mechanism of exchange. Details of the different CRISPR- cas loci were published separately. 13 One feature that has been reported to differ even between Synechocystis 6803 substrains is motility. Therefore, a standard motility assay was conducted and demonstrated that Synechocystis 6714 is non-motile (Fig. 4 ). However, among the known mutations that affect motility in Synechocystis 6803 substrains, we found an intact spkA protein kinase gene, 37 an intact hfq gene, 38 as well as most pil genes. 39 However, one missing gene in Synechocystis 6714 encodes an orthologue of PilA5 ( slr1928 in Synechocystis 6803), a type 4 pilin-like protein, which is involved in the formation of thick pili and motility 40 and therefore may explain the observed phenotype.\n Figure 4. Verification of physiological and genetic differences predicted upon draft genome analysis of Synechocystis 6714. (A) Phototactic motility of Synechocystis . Cells from liquid cultures (OD 750 = 0.2) were dropped onto a BG11-agar plate, pre-cultivated under standard conditions for 3 days and afterwards exposed to a gradient of incident light with intensity 50 µE. The photograph was taken before and after further 5 days. (B) Drop dilution assay showing the growth on solid media in the presence of increasing concentrations of Zn 2+ ions. The photograph was taken after 10 days of standard cultivation. Furthermore, a gene cassette involved in the sensing and the resistance to Zn 2+ and Co 2+ (including the genes corR, corT, ziaA, and ziaR ; Supplementary Table S5 ) 41 , 42 appears to be specific for Synechocystis 6803 and missing in strain 6714. The functional significance of this difference was tested in growth experiments in the presence of increasing amounts of Zn 2+ ions and revealed the higher tolerance of strain 6803 against high Zn 2+ levels (Fig. 4 ). Another four genes, which were not found in the Synechocystis 6714 genome, are the ggtABCD genes encoding a transport system for the (re-)uptake of the compatible solute glucosylglycerol. 43 , 44 Apart from that, the loci adjacent to ggtA or ggtBCD in Synechocystis 6803 are conserved in the genome of Synechocystis 6714 (Fig. 5 A and B). To verify the absence of Ggt, Northern hybridization with 32 P-labelled probes specific for ggtA or ggtBCD was performed with RNA from salt-treated cells. As expected, no mRNA was detected in salt-treated cells of strain 6714, whereas the expression level of the ggt genes correlated with the external salinity in Synechocystis 6803 (Fig. 5 C). Moreover, the relative abundance of the mRNA for ggpS , the gene encoding the key enzyme of glucosylglycycerol synthesis, the main compatible solute in these two strains, was measured and revealed its salt-dependent expression in Synechocystis 6714 (Fig. 5 C), similar to the well-characterized situation in Synechocystis 6803. 45 These results further substantiated that, even though the 6714 genome is not completely finished, the lack of certain genes correlates to physiological differences.\n Figure 5. Comparative genome analysis reveals the absence of the genes encoding the glucosylglycerol transport system (Ggt). (A) Genomic region encompassing the ggtA gene in Synechocystis 6803 and of the corresponding region in Synechocystis 6714. (B) Genomic region encompassing the ggtBCD operon. Apart from Ggt, both loci are well conserved (protein identity scores of ca. 90%). (C) Occurrence of mRNAs for ggtA , ggtBCD , and ggpS in salt-treated cells of both strains. For ggpS , salt-dependent expression was observed in both strains, whereas mRNAs for ggtA and ggtBCD were not detected in strain 6714. Deletion of Ggt in Synechocystis 6803 results in the inability of taking up GG as well as trehalose and sucrose. 43 , 44 Furthermore, the ggtA mutant of strain 6803 became leaky for GG, i.e. an increase in GG in the medium was observed when cells were grown in salt medium, suggesting that its transport is mainly necessary for recovery of GG leaked through the cytoplasmic membrane into the periplasm. 43 Due to the absence of Ggt in strain 6714, an uptake of GG seemed unlikely and a GG accumulation in the medium during growth at elevated salinities should be measureable. To test this hypothesis, the intra- as well as extracellular GG contents were measured for cultures acclimated to different salinities. Under freshwater conditions (0% NaCl), the cells of both strains were virtually free of GG. In Synechocystis 6803, the intracellular GG level increased corresponding to the external salt level, whereas in the surrounding medium, virtually no GG was found (Fig. 6 A). In principle, a correlation of the internal GG content and the external salt concentration was also observed for strain 6714. Up to a salinity of 4% NaCl, the GG concentrations with respect to the average biomass (expressed as OD 750 ) were similar. However, no further increase was observed when cells were grown at 6% NaCl pointing to a somewhat lower salt tolerance of strain 6714 (see below). Interestingly, GG also accumulated in high amounts in the surrounding medium, which supports the assumption that an effective system for the re-uptake of GG is missing in Synechocystis 6714 (Fig. 6 A). Similar to the internal, also the external GG content increased according to the salinity (Fig. 6 A).\n Figure 6. Effects of the absence or presence of the ggtABCD system. (A) Measurement of intracellular and extracellular GG content in the two Synechocystis strains. (B) Long-term growth of Synechocystis 6803 and 6714 in liquid cultures under salt stress. Cells were pre-cultivated without salt for 2 days before salt was added to a final concentration of 2, 4, and 6% (w/v), respectively (time point is marked by a red arrow). After further 4 days, samples for RNA extraction and GG measurements were taken (marked by a black arrow). The data are representatives of two independent experiments. (C) Drop dilution assay illustrating the growth on solid media in the presence of increasing NaCl concentrations. Cell material of exponentially growing cultures was diluted to an OD 750 of 0.3 and 20 µl of this suspension as well as a dilution series were dropped on NaCl-containing, agar-solidified BG11 medium. The photograph was taken after 10 days under constant illumination of 50–60 µE. The synthesis of GG is costly regarding the consumption of energy and carbon. Thus, an effective uptake system seems reasonable for a bacterium whose osmotic adaptation is based on GG accumulation. For a Ggt mutant of Synechocystis 6803, it was postulated that the inability to take up leaked GG should result in a lower salt tolerance or at least to a lower growth performance under higher salinities, especially if the cells are grown under Ci-limitation. 43 Interestingly, in liquid cultures that had a rather low surface:volume ratio, which results in a poor aeration in turn leading to a low degree of Ci availability, strain 6714 grew slower compared with strain 6803 in the presence of increased NaCl concentrations (Fig. 6 B). In contrast, both strains showed similar growth performance under freshwater conditions (0% NaCl). Moreover, strain 6714 also showed a lower salt tolerance when cells were grown on solid medium in the presence of various NaCl concentrations (Fig. 6 C). In the presence of 3% NaCl, no colonies were observed for strain 6714, whereas 6803 grew well under the same condition.\n\n4. Discussion The here presented draft genome sequence of Synechocystis 6714 allows comparative genome-based studies, as we demonstrate for several examples of physiological importance. Other comparative analyses include the direct comparison of promoter elements and of conserved sRNAs with similar regulation, implying conservation of function as we are showing in a separate manuscript. We have noticed several important differences between the two strains. As the absence of a gene from a draft genome sequence might be considered ambiguous, we have highlighted cases for which the physiological difference predicted by the lack of certain genes could indeed be demonstrated. Among these differences is the lack of a transport system for the re-uptake of the compatible solute glucosylglycerol, linked to the observation that strain 6714 showed growth retardation at salinities above 2%, whereas strain 6803 even managed 4% in liquid cultures. The accumulation of GG in the external medium meaning a permanent loss of fixed carbon might be reasonable for the reduced salt tolerance of strain 6714 as has been postulated earlier. 43 In addition to compatible solute accumulation, a balancing of the ionic composition is also important to cope with changing salinities. For instance, an active extrusion of Na + is essential for cyanobacteria in order to maintain a low, non-toxic intracellular level. Homologues for most genes known to be involved in Na + transport and which might be also important during salt acclimation (for review, see Hagemann) 46 are found in the genome of Synechocystis 6714. However, a homologue of sll1685 (PxcA) which might be involved in the energetization of Na + transport is missing. Furthermore, the genome of Synechocystis 6714 harbours two copies of the kdp operon each encoding a high-affinity K + transporter (genes sly5000010–sly5000050 and sly1021590– sly1021630 ), whereas strain 6803 has a single copy of this operon (slr1728–slr1731 ). The Kdp ATPase system, initially characterized in Escherichia coli , is responsible for the immediate uptake of K + after salt or osmotic shock in E. coli . 35 In combination with glutamate as an organic counter ion, K + is believed to act as a temporary compatible solute and moreover as a regulatory signal for the initiation of subsequent acclimation processes, also in cyanobacteria. 46 , 47 Interestingly, the kinetics for the uptake of K + in cyanobacteria after salt shock was characterized for Synechocystis 6714. 48 A sudden osmotic shift by adding 500 mM NaCl was followed by a transient accumulation of K + which started within the first minutes, peaked at around 30–60 min and declined after 24 h to levels similar to non-shocked cells. The decrease in K + was accompanied by an accumulation of GG. The kinetics of a K + uptake have not been measured so far for Synechocystis 6803, but it might be a bit different from the process in Synechocystis 6714 due to the absence of a second kdp operon. Another interesting observation is the putative substitution of a gene cassette of ∼40 kb encoding several glycosyltransferases, transport proteins, and hypothetical proteins in the two strains. Together with the presence of some genes not found in any other cyanobacteria and the strongly reduced average GC % content in this region, this region is likely representing a genomic island. Physiologically and ecologically important genomic islands have been identified in several marine cyanobacteria. 2 , 3 , 49 , 50 Interestingly, glycosyltransferase and glycoside hydrolase gene families have also been found frequent in several of these cyanobacterial genomic islands. Therefore, the modification of cell surface polysaccharide and lipopolysaccharide biosynthesis by several of these enzymes, presumably allowing diversification of cell surface features appears central for this group of organisms. Such modification capacity is likely to be relevant in the avoidance of grazers and even more in the avoidance of bacteriophage infection. 51 In conclusion, the draft genome analysis of Synechocystis 6714 allows to follow interesting research problems in this strain. However, most importantly, it opens exciting new opportunities when working with the most advanced cyanobacterial model, Synechocystis 6803."
} | 7,812 |
37662195 | PMC10473689 | pmc | 6,102 | {
"abstract": "Disturbance events can impact ecological community dynamics. Understanding how communities respond to disturbances, and how those responses can vary, is a challenge in microbial ecology. In this study, we grew a previously enriched specialized microbial community on either cellulose or glucose as a sole carbon source, and subjected them to one of five different disturbance regimes of varying frequencies ranging from low to high. Using 16S rRNA gene amplicon sequencing, we show that community structure is largely driven by substrate, but disturbance frequency affects community composition and successional dynamics. When grown on cellulose, bacteria in the genera Cellvibrio , Lacunisphaera , and Asticaccacaulis are the most abundant microbes. However, Lacunisphaera is only abundant in the lower disturbance frequency treatments, while Asticaccaulis is more abundant in the highest disturbance frequency treatment. When grown on glucose, the most abundant microbes are two Pseudomonas sequence variants, and a Cohnella sequence variant that is only abundant in the highest disturbance frequency treatment. Communities grown on cellulose exhibited a greater range of diversity (0.67–1.99 Shannon diversity and 1.38–5.25 Inverse Simpson diversity) that peak at the intermediate disturbance frequency treatment, or 1 disturbance every 3 days. Communities grown on glucose, however, ranged from 0.49–1.43 Shannon diversity and 1.37– 3.52 Inverse Simpson with peak diversity at the greatest disturbance frequency treatment. These results demonstrate that the dynamics of a microbial community can vary depending on substrate and the disturbance frequency, and may potentially explain the variety of diversity-disturbance relationships observed in microbial ecosystems.",
"conclusion": "Conclusion Here, we have demonstrated that communities will respond differently to the same disturbance regime, when grown on substrates of varying complexity. We observe a unimodal DDR when communities are grown on cellulose, a recalcitrant substrate. When grown on glucose, however, we observed a monotonically increasing DDR. Although substrate is a strong predictor for community composition, communities further cluster by disturbance frequency, and successional dynamics differ between disturbance treatments for the same substrate. These results suggest that the range of DDRs we observe across different microbial systems may be due to the nutritional resources microbial communities can access and the interactions between bacteria and their environment.",
"introduction": "Introduction Disturbance ecology investigates foundational questions of how systems and organisms respond to changing environments. Traditionally, disturbances are defined as discrete events that remove biomass directly or indirectly through displacement or mortality 1 , 2 . Fires, floods, and volcanic eruptions are classic examples of disturbances that change community composition by directly impacting species or altering the environment 3 , 4 . Early theoretical consideration of disturbance on community ecology include the Intermediate Disturbance Hypothesis (IDH), which predicts that the diversity-disturbance relationship (DDR) follows a “hump-backed”, or unimodal curve 5 . Support for the IDH has been mixed. Experimental measurements of the DDR for different systems has revealed a variety of trends, including both positive and negative monotonic, unimodal, bimodal, and several nonsignificant DDRs 6 . Recent frameworks of disturbance theory accommodate vastly different spatiotemporal scales between systems, and disentangle disturbance events and impacts 7 , 8 . The advent of high-throughput sequencing has widened the scope of questions that microbial ecology can ask, including research that investigates how disturbance impacts microbial communities. Researchers have studied disturbances in several different systems including marine sediment 9 , soil bacterial 10 and soil fungal communities 11 , and wastewater communities 12 . Microbial systems also display a variety of DDRs, which suggests that rather than trying to support or reject specific DDRs, researchers can better understand disturbance ecology by investigating the underlying factors that lead to different DDRs. Given the vast differences in systems between these studies, it is difficult to determine what specific factors lead to differing responses to a disturbance. Although we know that microbial community responses to disturbances can vary, whether the same community can exhibit different responses to the same disturbance and what factors would cause those differences, is relatively underexplored. Moreover, understanding what factors influence responses to a disturbance event is important for predictive power in studying microbial communities. To address this gap in knowledge, we examined the effects of disturbance on a bacterial community enriched from the refuse pile of the leaf-cutter ant Atta colombica that had previously been passaged in the lab on minimal media and cellulose by Lewin et al. 13 , 14 . Leaf-cutter ant refuse piles are composed of discarded plant biomass that has been partially degraded by the ants’ mutualistic fungal cultivar, Leucoagaricus gongylophorus \n 15 . Previous work has demonstrated that these refuse piles are enriched with plant-biomass degrading microbes 16 , 17 . Focusing on bacterial communities derived from leaf-cutter ant refuse piles, Lewin et al. experimentally evolved cellulose-degrading bacterial communities and investigated their compositional dynamics and cellulolytic abilities 13 , 14 . During each passage, a portion of the community was aliquoted into a new test tube containing fresh minimal media and a new strip of cellulose. These serial transfer events are analogous to disturbance events, as it is a species-independent method of biomass reduction and provides the “survivors” with a replenished ecosystem. This method of proxying disturbance through removing cells has been used in other studies 18 . Lewin’s community was enriched on cellulose, a recalcitrant crystal of β-1,4-linked glucose molecules. Cellulose is insoluble in water, and must be cleaned into cellobiose or glucose in order to be transported into a cell. Cellulase genes have limited distribution in bacteria, but β -glucosidases, which cleave cellobiose into glucose, are more widespread 19 . Thus, cellulolytic and noncellulolytic microbes compete for cellobiose – and these interactions may impact the community’s composition 20 , 21 . Lewin et al. 2022 evaluated successional dynamics in this microcosm by measuring the relative abundance of 16S rRNA genes every day for a week and found that a Cellvibrio operational taxonomic unit (OTU) was more abundant up to 48–72 hours, before other OTUs became more abundant 14 . This finding suggests that a cellulose degrader must proliferate and produce cellulases before noncellulolytic opportunists can take advantage of liberated cellobiose or metabolic byproducts. Our goal was to understand how substrate complexity interacts with disturbance frequency to shape community diversity. We hypothesize that diversity maximizes on a simple substrate (glucose) at higher disturbance frequencies but maximizes on a complex substrate (cellulose) at lower disturbance frequencies. We reasoned that on a simple substrate with low disturbance, competition exclusion would be a stronger driving force for community assembly while more frequent disturbances would disrupt competitive microbes from establishing. Conversely, on complex substrates, the ability to use the substrate would be a more important driving force. Since cellulases are phylogenetically limited in distribution 19 , we hypothesize that the bacteria that initially grow will be those that can degrade cellulose similar to what Lewin et al. 2022 observed 14 . Frequent disturbances should select for bacteria that can directly use cellulose. As cellulose is degraded into cellobiose, those molecules enrich the surrounding media and feed non-degraders. Thus, at infrequent disturbances non-degraders can grow making the community taxonomically richer. To test our hypotheses that diversity maximizes on glucose at high disturbance frequencies and maximizes on cellulose at lower disturbance frequencies, we subjected Lewin et al.’s cellulose-enriched community to two substrate treatments: minimal media supplemented with either glucose or cellulose. Each substrate was then subjected to five disturbance frequencies: passage every 1, 2, 3, 5, or 7 days. At the end of their assigned disturbance regime, we expanded the communities into multiple tubes of their respective substrate and destructively sampled over the course of one week. We then extracted DNA from these samples for 16S rRNA gene amplicon Illumina-based sequencing. Next, we analyzed these sequences to determine community composition and measured diversity. By comparing the same disturbance frequency between substrate complexities, we can evaluate how community diversity is affected by the interaction between disturbances and resources.",
"discussion": "Discussion In this study, we aimed to address how substrate and disturbance frequency interact to shape microbial community structure. We found that substrate is a main driver of the communities’ response to disturbance. We demonstrated that diversity peaks at the intermediate disturbance frequency, 1/3 days, when the community is grown on cellulose, a recalcitrant carbon source. However, community diversity peaks at the highest disturbance frequency, 1/7 days, when the community is grown on glucose, a labile carbon source. The results of this work show how community response to disturbances can be impacted by the substrate they are grown in and contributes to our understanding of how environmental factors interact with disturbances to impact bacterial communities. Diversity-Disturbance Relationships Our community displays a different Diversity-Disturbance Relationship (DDR) depending on the substrate it is grown on. When grown on cellulose, the community displays a unimodal curve ( Fig. 2 ) that fits with predictions of the Intermediate Disturbance Hypothesis, which posits that diversity peaks at intermediate disturbances 5 . However, the IDH has been found to be an inadequate framework as studies across a variety of ecosystems have found many divergent types of DDRs 6 , 31 . Our findings also demonstrate the incompleteness of the IDH; communities grown on glucose display a non-unimodal DDR. Differing DDRs resulting from the same experimental system have been observed before 18 . Hall et al. 2012 manipulated disturbance intensity (the proportion of cells they moved) and used a simpler one-species community – exploiting the ability of Pseudomonas fluorescens to exhibit distinct morphotypes based on access to oxygen. They found a flat, monotonically increasing, or unimodal DDR depending on the disturbance intensity 18 . Other experiments have found a variety of DDRs, including a U-shaped DDR 32 . A model of a two-member community, based on experimental observations, consistently found unimodal DDR, although the exact shape changed with time 33 . A more recent model of a two-member community displayed multimodality 34 . One potential reason we did not observe a unimodal DDR with our glucose treatment could be because we did not have a disturbance regime that was frequent enough to result in a population bottleneck. If the disturbances were so frequent that no, or very few, microbes were being passaged each time, then we might expect the diversity of the glucose communities to decrease. Disturbance disrupts community composition Following their assigned disturbance regime treatment, we sampled our experimental communities over the course of a week to evaluate how disturbance frequencies may impact community assembly. In the intermediate disturbance frequency for cellulose treatment (1/3 days), Cellvibrio is typically abundant, before being replaced by other taxa. This succession resembles what Lewin et al. 2022 reported. However, at lower frequencies (1/7 and 1/5 days) Lacunisphaera was also found to be abundant, and at higher frequencies (1/2 days and 1/1 days) Asticcacaulis increases in relative abundance. Notably, Cellvibrio starts at lower relative abundance before increasing in the high frequency disturbance treatments (1/2 and 1/3 days). It is important to note that Lacunisphaera was not identified in Lewin’s work. Lacunisphaera spp., which belong to the phylum Verrucomicrobia phylum do not have any reported have cellulolytic activity, although one isolate has been described to use a variety of carbon sources 35 . An Asticcacaulis ASV was abundant in our high frequency cellulose treatments and an Asticcacaulis OTU was found in Lewin et al. 2016 13 . Asticaccaulis has been found in other lignocellulolytic communities including communities derived from wood or forest soil 36 , 37 . Communities grown in glucose did not display obvious assembly patterns at most disturbance frequencies. We identified two abundant Pseudomonas ASVs in the glucose substrate treatments. We cannot determine if these represent different populations, but they appear to have different dynamics across disturbance frequencies. As our study was limited to 16S rRNA gene amplicon sequencing, we cannot determine what mechanisms led to the abundance of Pseudomonas ASVs in the glucose samples. Pseudomonas is a common environmental microbe, known best as a soil-dweller or member of the rhizosphere microbiome 38 . As enteric bacteria, the Pseudomonas ASVs likely have faster growth rates than other ASVs in these communities. Enrichment for copiotrophs when growth substrate is supplemented with labile carbon has been observed in a previous study 39 . Additionally, Pseudomonas are known for producing a variety of natural products, including molecules that suppress competing microbes 40 , 41 . This may be one explanation for how it came to dominate the glucose samples. The two Psuedomonas ASVs dominated community composition in most disturbance frequencies for communities grown in glucose, except for the highest frequency treatment which also had highly abundant Cohnella ASV. An isolate from this genus has shown cellulolytic ability 42 , 43 . Although we cannot explain why it is abundant in the high frequency glucose samples, the same ASV is also found in the intermediate frequency of our cellulose samples, which matches the report of Lewin et al. 22 that found Cohnella to be positively associated with cellulose degradation 14 . The different DDRs and successional patterns we observe are likely due to the differing interactions between microbes in the two substrates we considered. Cellulose is a recalcitrant substrate that must be cleaved into cellobiose (a glucose dimer) which is transported into the cell before being cleaved into glucose 44 . Cellvibrio is likely the dominant cellulose-degrader in this microcosm 13 , 14 . In order to degrade cellulose, Cellvibrio produces extracellular endoglucanases and exoglucanases that liberate cellobiose from the cellulose polymer 45 . Excess cellobiose molecules are likely what feeds the remaining community. Thus, non-cellulolytic organisms cannot immediately consume carbon in our cellulose treatments and must wait for cellulose-degraders to enrich the media with labile carbon. In contrast, glucose is labile, and thus competition is likely a much stronger driving force in community dynamics. Given that we used 16S rRNA gene amplicon sequencing, we cannot make definitive conclusions about the type of interactions in our community. However, substrate complexity is known to influence bacterial interactions. For example, a synergistic interaction found in co-cocultures of Citrobacter freundii and Sphingobacterium miltivorum on carboxymethyl-cellulose, xylan, lignin or wheat straw was lost when the pair was grown on glucose 46 . Conclusion Here, we have demonstrated that communities will respond differently to the same disturbance regime, when grown on substrates of varying complexity. We observe a unimodal DDR when communities are grown on cellulose, a recalcitrant substrate. When grown on glucose, however, we observed a monotonically increasing DDR. Although substrate is a strong predictor for community composition, communities further cluster by disturbance frequency, and successional dynamics differ between disturbance treatments for the same substrate. These results suggest that the range of DDRs we observe across different microbial systems may be due to the nutritional resources microbial communities can access and the interactions between bacteria and their environment."
} | 4,199 |
35519388 | PMC9065546 | pmc | 6,106 | {
"abstract": "The great potential of bioelectrochemical systems (BESs) in pollution control combined with energy recovery has attracted increasing attention. Classified by their functions in the BES, microorganisms including degraders, electricigens, and element cycle-related microbes play key roles in pollutant degradation and electricity generation, and the functions of these microbes are affected by various environmental and operating conditions. This review systematically summarizes the effects of crucial conditions on the efficiency of the process of contaminant removal combined with electricity generation in BESs, with particular focus on the pH, temperature, conductivity, substrates, inoculums, magnetic field and reactor design parameters, such as architecture, electrode material, and electrode potential. The aim of this review is to help reveal the microbial functions during the bioelectrochemical remediation of environmental media and to optimize the system by determining the appropriate conditions for functional microorganisms, thus better promoting the transition of BESs from the laboratory to actual applications.",
"conclusion": "5. Conclusion The BES must be robust enough to be applied in the field for bioremediation or energy production, and research is needed to investigate the functional microorganisms under the influence of various conditions. 29 It is difficult to clearly study the effects of various interaction measures on the microbial community structure, and yet such research makes sense for the exploration of the microbiological function mechanism and the optimization of BESs to better guide its transition from the laboratory to actual applications. Here, we summarize the effects of various factors on functional microorganisms and contaminant removal in BESs and provide corresponding optimization measures. Functional microorganisms in BESs, including electrogenic bacteria, degrading bacteria, and element cycle-related bacteria, harvest electrons from the degradation of substrates and promote the removal of contaminants, and these communities are affected by various environmental conditions. Here, we systematically summarized the effects of various enhancements (operational factors) on the BES performance with respect to the functional microorganisms and contaminant removal, with the aim of determining the optimal conditions during polluted environmental remediation by BESs and the effective measures that should be taken. Admittedly, the potential of BESs to remediate contaminated substrates needs to be further explored, and there are still some challenges yet to overcome in the use of BESs to actually remove contaminants. Therefore, the BES mechanism should be studied more deeply and comprehensively. At present, research on microorganisms in BESs remains mainly at the species identification stage, and we should therefore pay more attention to the specific roles of functional genes and functional proteins under particular conditions of the system. The study of electrogenic microorganisms is still an area worthy of attention, and the study of electrogenic microbial secretions and the biological interaction with coexisting neighbours involved in carbon, nitrogen, phosphorus and sulphur transformation will lead to new discoveries. Undoubtedly, the great potential for BESs in pollution control and energy recovery requires us to be committed to its practical application in the future.",
"introduction": "1. Introduction A bioelectrochemical system (BES) is a tool that converts chemical energy directly into a valuable resource, such as hydrogen in microbial electrolysis cells (MECs) and electricity in microbial fuel cells (MFCs), by means of microbial catalysis. 1–3 Presently, BESs show great potential for the removal of pollutants from a variety of environments, such as wastewater, 4–6 contaminated sediments 7,8 and soils. 9–12 BESs can be fed any biodegradable organic matter, from simple molecules (such as carbohydrates or proteins) to complex mixtures (such as petroleum hydrocarbons or swine wastewater), that can be effectively degraded by microorganisms in the system, resulting in the output of electrical energy or hydrogen. 13,14 Consequently, BESs reduce energy loss and waste generation, 15 which greatly reduces costs compared to physical and chemical remediation techniques. Furthermore, based on the inexhaustible electron acceptor of the solid anode, the enhancement ability of the biocurrent is sustained, which overcomes the deficiency of the electron acceptor in a contaminated medium. 16,17 However, there are still some problems to be solved in the actual application of BESs, such as improving the degradation of complex compounds, controlling the reaction process of microorganisms, and continuing to reduce costs. Recently, researchers have made extensive efforts to overcome some deficiencies, such as improving the reactor configuration and electrode materials, 18,19 finding efficient electrogenic bacteria, 20 and achieving multi-factor enhancement of BES. 21–24 In BESs, microorganisms play a key role in the degradation of pollutants and the generation of electricity or hydrogen, 25–27 so the intricate interactions of functional microbes have been thoroughly studied. However, the activity and number of functional microorganisms are directly influenced by the surrounding conditions. In this review, factors presently affecting the performance of BESs are summarized, e.g. , pH, temperature and conductivity. The understanding of relationships between the reinforcement measures and performance of a BES aims to assist in revealing the biological mechanism and providing the guidance for BES application."
} | 1,421 |
36838264 | PMC9964534 | pmc | 6,108 | {
"abstract": "It is claimed that one g of soil holds ten billion bacteria representing thousands of distinct species. These bacteria play key roles in the regulation of terrestrial carbon dynamics, nutrient cycles, and plant productivity. Despite the overwhelming diversity of bacteria, most bacterial species remain largely unknown. Here, we used an oligotrophic medium to isolate novel soil bacteria for positive interaction with soybean. Strictly 22 species of bacteria from the soybean rhizosphere were selected. These isolates encompass ten genera ( Kosakonia , Microbacterium , Mycobacterium , Methylobacterium , Monashia , Novosphingobium , Pandoraea , Anthrobacter , Stenotrophomonas , and Rhizobium ) and have potential as novel species. Furthermore, the novel bacterial species exhibited plant growth-promoting traits in vitro and enhanced soybean growth under drought stress in a greenhouse experiment. We also reported the draft genome sequences of Kosakonia sp. strain SOY2 and Agrobacterium sp. strain SOY23. Along with our analysis of 169 publicly available genomes for the genera reported here, we demonstrated that these bacteria have a repertoire of genes encoding plant growth-promoting proteins and secondary metabolite biosynthetic gene clusters that directly affect plant growth. Taken together, our findings allow the identification novel soil bacteria, paving the way for their application in crop production.",
"conclusion": "5. Conclusions This work adds to the inclusion of new species with significant potential for promoting plant growth. Taken together, here we demonstrated novel soil bacteria with a growth-promoting capability that had not previously been reported for soybean. In addition, we demonstrated the importance of coupling a more complex medium culture with bioinformatics approaches to select new PGPR. The findings enable the identification of distinct bacteria with a high potential for promoting plant growth, opening the path for future research and uses in agriculture intending to reduce the environmental impact of synthetic industrial pesticides and fertilizers, and to help mitigate drought stress.",
"introduction": "1. Introduction Plants are intimately intertwined with microbial communities in which several distinct mechanisms mediate dynamic ecological interactions. Plants release photosynthates belowground through mucilage and exudates, which are used as energy sources by distinct dwelling microbial taxa Berg [ 1 , 2 , 3 ]. In return, some specific microbial taxa can promote plant growth and/or offer protection against biotic and abiotic stressors. They occur via the synthesis of phytohormones, acquisition of nutrients, and antagonistic interactions with plant pathogens. It is estimated that less than 1% of bacterial species have been cultivated under laboratory conditions, a phenomenon known as the “Great Plate Count Anomaly” [ 4 ]. Soils are by far the richest environment, containing an extensive and diverse set of bacteria, of which the majority are yet unknown and are mainly detected by metagenome analysis [ 5 , 6 ]. Hence, the ecological features of most soil bacterial taxa, including their environmental preferences, phenotypes, and metabolic capacities are mostly unknown. Apart from the rhizosphere, most of the soil is considered an oligotrophic environment. This area is distinguished by lower levels of microbial density and activity than those in high-resource environments [ 7 ]. The microbiome that inhabits this habitat is classified as a k-strategist, which implies that it can survive in low-nutrient conditions, grow at a slower rate, and has a high tolerance to toxic compounds [ 8 , 9 ]. Yet, most of the cultivation methods used in microbiology rely on nutrient-rich media, which may limit the study of oligotrophic bacteria from soil ecosystems [ 10 ]. These bacteria play key roles in regulating terrestrial carbon dynamics, nutrient cycles, and plant productivity. Therefore, novel strategies for assessing this unknown biological diversity are necessary. Studies have shown the potential for cultivation of previously ‘unculturable’ bacteria from environmental samples using simple cultivation strategies [ 11 , 12 ]. The cultivation of ‘unculturable’ bacteria can be improved by combining oligotrophic media, extended incubation periods, and selection of slow-growing bacteria [ 12 , 13 ]. The goal of this study was to isolate and identify novel microbial species with the potential to plant growth-promoting rhizobacteria (PGPR) for soybean plants, as well as to apply genomics approaches to gain insights into bacteria–plant interactions.",
"discussion": "4. Discussion In this study, 22 soybean growth-promoting rhizobacteria were identified. A total of 78% of the 22 bacteria isolated exhibited positive results for water stress growth, indicating their potential for application in crops with a shortage of water. The isolates produced IAA, the primary auxin found in plants, which is synthesized in the apical system of the stem and transported to the roots; plant-associated microbes can also synthesize it. Its primary effect is the growth of roots and stems [ 40 , 41 ]. An in vitro test of soybean revealed this elongation effect. Furthermore, the isolates were able to solubilize phosphorus, an important element in plant metabolism. Plant growth is hampered by its absence; nevertheless, it is the least available nutrient for the plant, since it is held by the precipitation of other soil elements, resulting in insoluble inorganic phosphates with the lowest available for the plant [ 42 , 43 ]. As a result, bacteria with the capacity to solubilize insoluble inorganic phosphate sources, increasing the soluble phosphorus level in the soil solution, and plant availability, play a crucial role in the phosphorus biogeochemical cycle [ 44 ]. The genera Bacillus , Pseudomonas , and Burkholderia have been commonly identified as widely prevalent growth-promoting bacteria in soybeans [ 45 , 46 , 47 ]. Here, by contrast, we discovered Kosakania sp., Microbacterium sp., Mycobacterium sp., Methylobacterium sp., Novosphingobium sp., Anthrobacter sp., Stenotrophomonas sp., Monashia sp., and Pandoraea sp. This could be due to the VL55 isolation medium used in this study. VL55 is a defined medium that is low in nutrients and has pH in the most acidic range. Because xylan is the only available carbon source, it is commonly employed for the selection of bacteria that were previously classified as “unculturable” in soil [ 11 , 48 ]. Although the isolates identified in this study were grouped close to the aforementioned genera, phylogenetic analysis revealed that they may belong to new genera and/or species. Some of the genera shown here are related to plant growth promotion activities, such as Microbacterium in neem growth promotion [ 49 ]. Another example is the genus Methylobacterium , which absorbs carbon molecules during endophytic associations and releases biopolymers, organic acids, coenzymes, vitamins, and toxins that aid in disease management [ 50 , 51 ]. Yet, few studies have been conducted to investigate the interactions of this genus with plants. In addition to species already known to promote plant growth, genera such as Novosphingobium are known to produce enzymes capable of degrading aromatic compounds [ 52 , 53 ]; Arthrobacter is used in many industrial applications and has the potential to be used in bioremediation [ 54 ]. Surprisingly, genera known to cause diseases in humans, such as Pandoraea and Stenotrophomonas , were isolated. However, based on the phylogenetic distance between the species, these isolates may belong to a distinct group of bacteria unrelated to human diseases. Microbacterium sp. strain SOY4 and Monashia sp. strain SOY12 were effective in suppressing the phytopathogen Fusarium oxysporum f. sp. phaseoli . Both isolates were Actinobacteria, a phylum recognized for producing secondary metabolic metabolites with broad antifungal properties [ 55 ]. In addition, we have showed the ability of two isolates SOY2 ( Kosakonia sp.) and SOY23 ( Agrobacterium sp.) to enhance soybean growth under drought in greenhouse conditions. Plants seeds inoculated with these bacteria generated more dry matter in the shoots and had a smaller reduction in leaf area than the other treatments. Although Agrobacterium has been identified as a plant pathogenic bacterium, certain studies have shown that specific tumor-inducing Agrobacterium strains can stimulate plant growth in non-susceptible plant hosts [ 56 , 57 ]. The use of genomics techniques in investigations aimed at the potential of plant growth-promoting bacteria has provided evidence of the genetic characteristics that promote the microbe–plant interaction [ 58 ]. Here, we gained insight into the genomic potential of two bacteria of our isolates SOY2 and SOY23 along with the bacteria genus mentioned in this study. We confirmed that SOY2 and SOY23 did not belong to any of the nearest species and represented two novel bacteria species, and together with 169 genomes, they have a repertoire of genes encoding for plant growth-promoting proteins with direct effects on the plant, such as bio-fertilization, iron acquisition, P and K solubilization, phytohormone synthesis, and nitrogen fixation. In addition, secondary-metabolite BGCs analysis revealed a variety of novel secondary metabolite BGCs to be explored. Well-known plant growth-promoting rhizobacteria (PGPR) have been widely used in the commercial sector with success; however, the search for new microbes that are resilient to adverse effects such as drought and have the potential to promote the growth of crops plants is becoming increasingly relevant, given climate change and its impact on food production. Chauhan and colleagues [ 59 ] reported several novel PGPRs that have not yet been achieved for commercial scales of production."
} | 2,478 |
40092618 | PMC11910081 | pmc | 6,109 | {
"abstract": "Summary Anti-smudge coating materials have a broad prospect, but they are susceptible to wear from nails and sand. Therefore, the potential application of such coatings on glass substrates needs coating features such as superhardness and high transparency. However, realizing these key properties combined with anti-smudge function is significantly challenging. In this work, we show a conceptional nanoparticle pattern designing strategy of materials, inspired by stepping on cobblestone roads with the foot feeling of only the hardness of stones. Realize the nanoparticle pattern surface of “cobblestone roads” via facile and scalable interfacial reactions within a molecular compatible system, to successfully achieve the desired coating material properties including anti-smudge, superhardness, and high transparency. The coating was composed of tensely crosslinked sub-10 nm building blocks that bear an anti-smudge molecular layer, exhibiting undistinguished inorganic phase behavior when it was subjected to external forces within the contact point of micro- or above 10 nm nanoscale.",
"conclusion": "Conclusion In summary, we have created an anti-smudge coating with superhardness and high transparency via the nanoparticle pattern surface designing strategy of materials. Excellent repellency toward various liquids, hardness of above 9H, and transmittance of 98% were simultaneously achieved for this coating material. In addition, the chemicals and processes involved make this coating readily applied in large-area, daily, on-site applications on various substrates. The coating with combined properties is expected to have applications in fields including electronic screens and vehicle windows.",
"introduction": "Introduction Anti-smudge coatings with low-adhesive surfaces can protect substrates against both aqueous and oily contaminants, showing significant importance and promise for self-cleaning application scenarios. 1 , 2 , 3 , 4 , 5 , 6 Generally, the anti-smudge function of materials is achieved with low-surface-energy compounds enriched on the surfaces, such as fluorine- or silicon-based amphiphobic organic molecules or polymers. 7 , 8 , 9 , 10 , 11 The inherent softness of organic compositions makes materials with anti-smudge properties exhibit limited hardness, which are critical issues for the robustness of coating materials as well as the protection of various underlying substrates. 12 , 13 , 14 In practical applications, high hardness and transparency are important for the application of the coating, such as the coating used on screens of mobile phone screens, windows, and cars. The applied glass may face the wear of nails and sand, while the line of sight must be not affected during use. Furthermore, high hardness can prolong the coating’s lifetime, avoiding abrasion during the application. Thus, high hardness and transparency of anti-fouling coatings are essential to fulfill the applicable requirements. To improve hardness and scratch resistance, inorganic hard fillers have been incorporated into the anti-smudge organic coating materials. 15 , 16 , 17 , 18 However, these hybrid coating composites tend to have significant heterostructure, Young’s modulus mismatch, and weak interfacial interaction between inorganic fillers and organic matrixes, thus typically resulting in only modest improvements in hardness. Moreover, these fillers, and various nanomaterials that were used to fabricate superamphiphobic surfaces 19 , 20 , 21 or SLIPS, 22 as well as the crystallinity of PTFE-based materials tend to scatter light, which can affect the transparency of these materials 23 , 24 with anti-smudge performance and render them unsuitable for applications as wearable electronics or windows. To the best of our knowledge, only polyhedral oligomeric silsesquioxane (POSS), with cage-like inorganic/organic hybrid structure, was reported to achieve both anti-smudge function and superhardness (above 9H, inorganic level), and the molecular scaled composition can avoid the light scattering induced opacity. 25 , 26 , 27 However, the complex preparation, low solubility in various solvents, and high price of POSS restrict its large-scale industrial production as well as the application of hard anti-smudge coatings in different fields. 28 , 29 Inspired by stepping on cobblestone roads with the foot feeling of only the hardness of stones, herein, we proposed a conceptional strategy, a nanoparticle pattern surface based on ultra-small nanoparticles, to balance and guarantee the desired material properties including anti-smudge performance, superhardness, and transparency. Unlike the traditional method of choosing and integrating different organic/inorganic composites, our approach utilized interfacial reactions within a molecular compatible system, to realize the reduced-scale particle pattern of “cobblestone roads.” The building blocks function as ultra-small “cobblestones” with a size of only several nanometers to form the superhard particle pattern surface. Thus, after the coating is crosslinked, the coating could show the cobblestones road-like particle surface properties and external factors cannot affect the crosslinked part of the particles, mimicking a cobblestone road where hard cobblestones are wrapped in cement and the surface only shows the nature of cobblestones. In addition, interfacial molecular engineering endowed the building blocks with highly crosslinkable and anti-smudge functional segments. The resulting clear solution can be easily sprayed, dipped, or painted on-site onto various objects, and subsequently obtain the desired superhard, anti-smudge, also transparent coating materials. These coatings use inorganic nanoparticles to cover surfaces with the highest hardness requirements to avoid the problem of insufficient hardness of organic components, and the resulting transparent solution can be easily sprayed impregnated or applied on-site to various objects (such as aluminum, iron, glass, and polytetrafluoroethylene). Thus, these coatings are promising for various applications, such as electronic touch screens, windows of vehicles or skyscrapers. We believe that the nanoparticle pattern surface strategy can be used for the fabrication of many materials including those that wish to achieve the combination of different organic and inorganic characteristics.",
"discussion": "Results and discussion Preparation and characterization of the coating Our approach for preparing anti-smudge superhard transparent coatings is shown in Figure 1 A. Specifically, the superhard organic core of building blocks bearing amino groups was initially prepared by the hydrolyzation of 3-aminopropyl triethoxysilane (APTES). Subsequently, the core can react with the double bonds of the pentaerythritol triacrylate (PETA) comprised shell via Michael addition. Similarly, the amino groups of amino-terminated polydimethylsiloxane (NH 2 -PDMS-NH 2 ) can also react with PETA to impart the building blocks with anti-smudge properties, while the surplus double bonds of PETA would contribute to the highly crosslinking reaction among building blocks. These reactions resulted in a clear solution comprising the building blocks with the size primarily distributed between 1 and 10 nm ( Figure 1 B). Figure 1 Preparation and transparency (A) Illustration of the design and preparation of anti-smudge superhard transparent coating via “cobblestones road” nanoparticle pattern designing of materials. (B) The size distribution of the building blocks, and the picture indicating the crystal clearness of the aqueous solution containing the building blocks. (C and D) The SEM (C) and AFM (D) images of the coating surfaces, exhibiting the reduced-scale structures of cobblestone roads with a surface roughness (Ra: 0.441 nm). (E) Transmittance spectra of the coating, with the inserted picture indicating the transparency of the coated glass. This solution is applicable for industrial coating techniques such as spraying and painting, and the final coating materials could be obtained after curing at 180°C for 2 h. As predicted, the homogeneous morphology of the tensely crosslinked building blocks, exhibiting the reduced-scale structures of cobblestone roads, was observed via the SEM images ( Figure 1 C) and the atomic force microscopy (AFM) images ( Figure 1 D) with an average Ra of 0.484 ± 0.037 nm ( Figure S1 ), indicating the good smoothness of the coating. The energy dispersive x-ray (EDX) results also indicated a high level of element homogeneity of the coating ( Figure S2 ), and the element content was further analyzed via X-ray photoelectron spectroscopy (XPS) ( Figure S3 ). It is noteworthy that the curing temperature of coatings would initiate the remaining double bonds on the surfaces of building blocks thus obtaining the tense multi-crosslinking of the coating matrix. Furthermore, the spontaneous enrichment of the PDMS segments anchoring on the surface of the building blocks can create a low-surface-tension liquid-like monolayer for a long-lasting anti-smudge function. The transmittance of the coating was found to be above 98% in the entire testing range of 500–800 nm, and the insert picture of glass bearing the transparent coating does not obscure the vision ( Figure 1 E). This optical clarity would be attributed to the good distribution and sub-10 nm size of the building blocks, which are significantly smaller than the wavelength of visible light and would not cause undesirable scattering. 30 , 31 The chemicals and processes involved are readily available for the scalable preparation of this coating material. Liquid repellency properties of the coating To demonstrate the anti-smudge property of the coating, as shown in Figure 2 A, the contact angles (CAs) and sliding angles (SAs) of various liquids with different surface tensions (including n-hexane, ethanol, N, N -Dimethylformamide (DMF), hexadecane, diiodomethane, and water with surface tensions of 20.3, 22.1, 25.7, 27.2, 50.8, and 72.8 mN/m at 20°C, respectively) and various oils with different viscosities (including peanut oil, pump oil, and crude oil with viscosities of ∼80, 200, and 300 cP at 20°C, respectively) were evaluated. All these liquid droplets could easily slide off the coating surfaces, even with small CAs, which indicates the desired low adhesion and excellent anti-smudge performance of the coatings. Based on the CAs of water and n-hexadecane on the coated/uncoated surface, the surface energy of the coated surface was calculated to be approximately 22.72 mJ/m 2 , largely smaller than that of the uncoated one (55.75 mJ/m 2 ) (see Table S1 ). Photographs in Figure 2 B showed the processes of the water, hexadecane, and peanut oil sliding off the coating surfaces, these liquids would slide quickly without leaving any residue. Figure 2 Good antifouling properties of the coating (A) CAs and SAs of different liquids on the coating surfaces. (B) Photographs of various liquids sliding off the coated glasses without leaving any residue. (C) The image illustrating the liquid-like PDMS segments that anchor on the surface of the building blocks, thus achieving the anti-smudge surface function. (D) The coating maintained its liquid repellency and anti-smudge performance when it was subjected to local pressure resulting from two pieces of squeezed glasses, tested with water-based ink. (E) The sprayed oil-based graffiti can be readily wiped clean even via dry tissues. Lotus-inspired superhydrophobic or superoleophobic surfaces exhibiting liquid repellency rely on the entrapped air layer and surface topography and, thus would fail when in contact with pressured liquids as that tend to permutate into the textures and displace the air. 32 , 33 , 34 Unlike these pressure-sensitive rough surfaces, our coating was mainly attributed to the liquid-like anti-smudge segments that anchoring on the surface of the building blocks, 35 , 36 and the molecular functional surface is supposed to maintain its anti-smudge performance under pressure ( Figure 2 C). As shown in Figure 2 D and Video S1 , the ink liquids were dropped both onto the uncoated and coated areas of the glass surface, and the other glass (also with uncoated and coated regions) was then placed on top and pressed down matching underneath to provide the local pressure. The spread liquid on the uncoated/coated glass interface exhibited different wetting phenomena when removing the top glass. It was observed that the droplets on the uncoated area spread out and were seriously stained, while the droplets on the coated interface shrank quickly and reverted back to smaller droplets. In contrast, if using an original glass to press, the liquid on the coating would be adhered to and carried away by the upper uncoated glass, leaving a clean coating surface without ink liquid, as shown in Figure S4 . This indicates that when compared with the glass surface, the coating exhibited significantly lower adhesion properties. In addition, the coating exhibited easy clean properties toward undesired foreign materials such as graffiti. As shown in Figure 2 E, when the oily inks of Sharpie markers are sprayed onto the coated glass, the graffiti can be easily removed even with the use of dry tissues. However, the oily ink traces on uncoated glass were difficult to remove ( Figure S5 ). \n Video S1. Good liquid repellency Water-based ink shrank behaviors on the coating after pressure. \n Superhardness and good substrate adhesion of the coating To enhance hardness is challenging for liquid repellency surfaces with different mechanisms including Lotus-inspired textures 37 , 38 and Nepenthes-inspired liquid films. 39 , 40 In contrast, the superhardness >9H at the inorganic level of our coating was determined according to the standard ASTM D3363 protocol. 41 , 42 The test indicated the highest pencil hardness rating of >9H, with unnoticeable scratches when the coating was subjected to the scratching using a 9H pencil with a load of 1 kg and the ink could still shrink on the scratched coating ( Figure S6 ). This superhardness demonstrates superior performance compared to the commercial polyurethane (PU) coating, which exhibited a hardness (H) below the median score of the hardness scale ( Figure S7 ). To avoid the impact of the substrates on the coatings, an indentation depth of 500 nm corresponding to less than 1% of the coating thickness (∼60 μm, Figure S8 ) was used according to ISO 14577-4 standards. 43 , 44 Furthermore, the surface mechanical properties of the coating were further investigated via nanoindentation and nanoscratch tests ( Figure S9 ). The nanohardness and elastic modulus values of the coating obtained as average values from five measurements were 368.7 MPa and 4.68 GPa, respectively, and the experimental values are listed in Table S2 . For the wear test, the coating was abraded using steel wool under an average pressure of 15.0 kPa, and no peeling or scratches were observed on the coating surface after 200 abrasion cycles ( Figure S10 ). Although the CAs and SAs of water and hexadecane were slightly changed after various abrasion cycles ( Figure 3 A), the droplets of these liquids were still able to slide off the coating surface without leaving any residual traces ( Figure S11 and Video S2 ). The coating maintains anti-smudge properties after 1,000 cycles of abrasion when using cotton under an average pressure of 15.0 kPa Figure S12 shows the CAs and SAs of water and hexadecane after various abrasion cycles. Compared to other literature, 45 , 46 , 47 , 48 our coating exhibited well-combined features of antifouling, transmittance, and hardness, as shown in Figure S13 . Figure 3 Good stability of the coatings (A) The CAs and SAs of the coatings after abrasion using steel wool for various cycles. (B) The illustration of the superhard mechanism of the anti-smudge surface when the coating is subjected to external forces. A micro- or nano-scale tip would not distinguish the sub-10 nm blocks and cause scratches. (C) Shear strengths of coatings applied onto various substrates including metals and glass. (D) The coated and uncoated steel for the corrosion protection tests at room temperature (∼28°C), using HCl, CuSO 4 , and NaCl solutions as corrosive liquids. (E) Tafel polarization curves of bare and coated tin plates. (F) The CAs and SAs of the coatings after the ultrasonic treatment (40 K Hz). \n Video S2. Abrasion performance Water slid down form the coating after abrasion. \n The superhardness and excellent wear resistance mechanism of the anti-smudge surface are illustrated in Figure 3 B. Just like we can only feel the hardness of stones when our footsteps on cobblestone roads, for this tensely crosslinked matrix comprised the sub-10 nm blocks, a micro- or nano-scaled tip should not be able to distinguish the blocks from the crosslinked interfaces. Thus, this coating would act as a continuous inorganic phase behavior when it is subjected to external forces with a contact point of above 10 nm length scale. Contrary to the anti-smudge low adhesion surface of the coating, a strong adhesion bottom to substrates was achieved. As shown in Figure 3 C, the shear strengths of the coating attached to a range of substrates were evaluated, including the tin plate, stainless steel (403), Al, and glass. 49 Among them, the coating on Al exhibited the lowest shear strength, which may be attributed to the highest thermal expansion coefficient of Al (∼23.6 × 10 −6 /°C) 50 with significant surface expansion and shrinking changes. However, regardless of these substrates, a minimum shear strength higher than 200 kPa was achieved. This high level of adhesion could be attributed to the well-leveling and film-forming properties of the coating solution, resulting from the sub-10 nm composition and tense crosslinking chemistry. In addition, the chemical bonds could form between the hydroxyl group on the substrate surface and the residual silane agent of the coating. The hydroxyl and amino groups of the coating could form hydrogen interaction with the substrates. Thus, the coating is firmly attached to the metal substrate, and results in a strong adhesive bottom layer. The different shear strength values measured for various substrates can be ascribed to the varying substrate composition that resulted in different synergetic interfacial adhesion interactions between the coating materials and the substrates, e.g., hydrogen bonding, hydrophobic interactions, and van der Waals forces, etc . 51 Thus, the coating could be firmly attached to various hydrophilic and hydrophobic substrates, such as iron sheet, glass, porcelain, polylactic acid (PLA), and polytetrafluoroethylene (PTFE). A knife was used to create a hundred grid with an interval of 1 mm on the coating, and then adhered and peeled off via a 3M tape. The result showed that no species was peeled off after the adhesion strength test, indicating good adhesion between the coating and various substrates ( Figure S14 ). Corrosion resistance and good transparency To determine the corrosion resistance of this coating, the half-coated steel sheet was subjected to various corrosive liquids, including CuSO 4 (1.0 mol/L), NaCl (1.0 mol/L), and HCl (1.0 mol/L). As shown in Figure 3 D, the coated tin plate surface showed no signs of corrosion, while the uncoated area appeared to be seriously corroded. The anti-corrosion performances of a bare tin plate and a coated tin plate were further examined by an electrochemical corrosion test. As shown in Figure 3 E and Figure S15 , in comparison with the bare tin plate, the coated area exhibited a positive shift in the corrosion potential (Ecorr) and a reduction in the corrosion current density (Icorr). Generally, a higher Ecorr value and a lower corrosion Icorr value indicate that the coating provides better corrosion protection and is able to slow the dissolution rate of the substrate, which thus demonstrates that this coating imparts corrosion resistance. 52 Moreover, when the coating was immersed in various organic solvents (including n-hexane and N, N -dimethylacetamide (DMAC)), aqueous solutions with pH values ranging from 3 to 11, and artificial seawater for 21 days, the sliding behaviors of water and hexadecane on the coating surface were discovered negligible changes ( Figures S16 and S17 ). In addition, the coating also maintained its liquid repellency after the treatment of underwater ultrasonic waves for 180 min ( Figure 3 F). These results confirmed that the coating is highly resistant to damage and has excellent durability for providing long-term protection of substrates. It is noteworthy that the optical transparency of coating is important for the practical application of anti-smudge materials. Thus, this coating can be applied to the screen of electronic devices (e.g., smartphones) without changing its optical clarity ( Figure 4 A). The coating’s repellency toward an artificial fingerprint liquid (consisting of lactic acid, acetic acid, sodium chloride, sodium hydrogen phosphate, 1-methoxy-2-propanol, hydroxyl-group-terminated polydimethylsiloxane, and deionized water) was investigated, 53 and an aqueous ink solution (1 wt %) was added to the artificial fingerprint liquid to present a clearer enlarged image of the trace that was pressed by a finger on the coated ( Figure 4 B) and uncoated ( Figure S18 ) glass plates. The uncoated glass is readily wet and contaminated by the artificial fingerprint liquid. However, when this liquid is applied to the coated part, it contracts into distinct droplets immediately, and this contraction of the fingerprint would obviously weaken the disturbance toward reading. Figure 4 Potential application of the antifouling coatings (A) The application of coating on the screen of a smartphone. (B) The contraction of artificial fingerprint liquid on the coating surface after pressed by a finger, which were magnified via a microscope. (C) The application of the coating on the car windscreen and the droplets would slide downward in natural surroundings. (D) Force analysis of the raindrops on the windscreen, where “F,” “f,” “N,” and “α” represents the wind force, resistance, supporting force, and the windscreen angles, respectively. (E) The droplets would slide upward on a coated windscreen with wind conditions. (F) Sliding movements of the liquids with different volumes. This transparent coating could also find an application on the windscreen, as it repels liquid contaminations ( Figure 4 C and Video S3 ) as well as dust, which is readily carried away by rain ( Figure S19 ). Remarkably, this coating can improve driving safety during rainy weather, as reducing raindrop retention can prevent rain-induced poor visibility. Sliding or staying of raindrops that splatter on the windscreen of a moving car is influenced by both gravity and the wind force. 54 To simulate this situation, we use a hair dryer to blow on the model windscreen with various volumes of water droplets, thus conducting a force analysis on the motion of the droplet, combined with Newton’s second law “F − f − mgsinα = ma” ( Figure 4 D). The experimental results also indicated that the raindrops were pulled down by gravity when the mess exceeded a certain threshold, while smaller ones were pushed upward when the wind prevailed ( Figure 4 E and Video S4 ). At both the sliding down and upward circumstances, the windscreen was clear without vision-disturbed residue. In contrast, medium-sized and very little droplets would stay as the difference between gravity and wind force could not overcome the friction, and these droplets would obstruct the driver’s vision thus causing danger. Surprisingly, the volume range for droplets to stay was discovered to be very limited for the coated windscreen, while that is significantly larger for the uncoated glass. For a quantitative calculation according to the various liquid volumes, it was found that the liquid-solid contact fraction of the coating is only one-tenth of that of the uncoated glass. \n Video S3. Sliding performance Water slide down from the coating on the car windscreen. \n \n Video S4. Anti-droplets adhesion on car windscreen The droplets would slide upward on a coated windscreen with wind conditions. \n Conclusion In summary, we have created an anti-smudge coating with superhardness and high transparency via the nanoparticle pattern surface designing strategy of materials. Excellent repellency toward various liquids, hardness of above 9H, and transmittance of 98% were simultaneously achieved for this coating material. In addition, the chemicals and processes involved make this coating readily applied in large-area, daily, on-site applications on various substrates. The coating with combined properties is expected to have applications in fields including electronic screens and vehicle windows. Limitations of the study The limitation of this study is that the structure and roughness of the coating surface cannot be adjusted to prepare series patterns."
} | 6,265 |
38873164 | PMC11169877 | pmc | 6,110 | {
"abstract": "A comprehensive study was conducted in the Cuatro Ciénegas Basin (CCB) in Coahuila, Mexico, which is known for its diversity of microorganisms and unique physicochemical properties. The study focused on the “Archaean Domes” (AD) site in the CCB, which is characterized by an abundance of hypersaline, non-lithifying microbial mats. In AD, we analyzed the small domes and circular structures using metagenome assembly genomes (MAGs) with the aim of expanding our understanding of the prokaryotic tree of life by uncovering previously unreported lineages, as well as analyzing the diversity of bacteria and archaea in the CCB. A total of 325 MAGs were identified, including 48 Archaea and 277 Bacteria. Remarkably, 22 archaea and 104 bacteria could not be classified even at the genus level, highlighting the remarkable novel diversity of the CCB. Besides, AD site exhibited significant diversity at the phylum level, with Proteobacteria being the most abundant, followed by Desulfobacteria, Spirochaetes, Bacteroidetes, Nanoarchaeota, Halobacteriota, Cyanobacteria, Planctomycetota, Verrucomicrobiota, Actinomycetes and Chloroflexi. In Archaea, the monophyletic groups of MAGs belonged to the Archaeoglobi, Aenigmarchaeota, Candidate Nanoarchaeota, and Halobacteriota. Among Bacteria, monophyletic groups were also identified, including Spirochaetes, Proteobacteria, Planctomycetes, Actinobacteria, Verrucomicrobia, Bacteroidetes, Candidate Bipolaricaulota, Desulfobacteria, and Cyanobacteria. These monophyletic groups were possibly influenced by geographic isolation, as well as the extreme and fluctuating environmental conditions in the pond AD, such as stoichiometric imbalance of C:N:P of 122:42:1, fluctuating pH (5–9.8) and high salinity (5.28% to saturation).",
"conclusion": "5 Conclusion We described here 325 MAGs from the AD site, comprising both Archaea (48) and Bacteria (277), spanning remarkably 40 phyla across both domains. The AD site displays high salinity and fluctuating pH and has been of interest since its discovery in 2016 because of its unique physicochemical conditions that support the growth of extremophile organisms. Our study provides information on the remarkable diversity and unique characteristics of microorganisms at AD, and the MAGs reported here enhanced our understanding of the prokaryotic tree of life, revealing a diverse microbial community, which, viewed from a phylogenetic perspective, suggests that the AD site might harbor many endemic lineages. The study highlights the exceptional microbiological diversity in this environment, as none of the MAGs could be classified at the species level, and a significant portion (126 MAGs) could not be classified even at the genus level. These results strongly suggest the presence of previously unknown microbial species and genera at this site. Phylogenetic analysis also reveals monophyletic clustering patterns, which could suggest that microorganisms at the AD site are endemic to CCB. We consider that the collection of MAGs obtained in this study will serve as a valuable resource for expanding the knowledge of microbial diversity within the tree of life.",
"introduction": "1 Introduction In recent years, culture-independent techniques have revolutionized our understanding of microbial diversity and evolutionary relationships within the phylogenetic tree of life. Notably, the discovery of the Candidate Phyla Radiation (CPR group) by Hug et al. (2016) , and the novel archaeal phylum Lokiarchaeota by Spang et al. (2015) have had profound impacts on our knowledge of microbial taxonomy, greatly expanding the phylogenomic coverage of the tree of life. More recently, Gong et al. (2022) identified several novel bacterial phyla within the FCB superphylum, highlighting the importance of MAGs in uncovering previously unknown microbial lineages and their ecological roles. The Cuatro Ciénegas Basin (CCB) is in central Mexico, is located in the state of Coahuila, and provides a unique setting for exploring microbial diversity, spanning a valley measuring ≈30 km by 40 km at ≈740 m above sea level and is surrounded by high mountains (>3,000 m). The CCB is a closed evaporitic basin that receives ≈150 mm of annual precipitation. This basin is also characterized by its oligotrophic conditions, as ecological analyzes have revealed that a nitrogen-phosphorus ratio of 16:1 (the Redfield ratio) is common to most life on Earth ( Elser, 2006 ). In the CCB oasis, however, these ratios are skewed due to the low level of phosphorus in the ecosystem. For example, there is a very high ratio of nitrogen to phosphorus (167:1) in the sediment of the Churince hydrological system ( Souza et al., 2018 ). A trace of this evolutionary history has also been reported in the extreme imbalance at the bacterial intracellular level in many lineages (the most extreme being nitrogen to phosphorus ratio of 965:1 in a strain of CCB Bacillus cereus group) ( Valdivia-Anistro et al., 2016 ). Despite this extreme N:P unbalance, CCB harbors an extensive system of springs, streams, and ponds of significant scientific interest and is thought to have “the highest level of endemic biodiversity in all of North America,” at least based on macroscopic organisms (70 endemic species within 500 km2) ( Stein et al., 2000 ; Souza et al., 2006 , 2012 ). On the other hand, the nitrogen-phosphorus (N:P) ratio in the CCB can vary widely, from conditions of severe phosphorus deficiency to near-normal conditions, which has a direct impact on microbial proliferation ( Elser et al., 2000 ). This environment has triggered evolutionary responses in endemic microorganisms, such as, the reduction of the genome of Bacillus coahuilensis, and its production of sulfolipids instead of phospholipids as potential adaptations to low phosphorus concentrations ( Alcaraz et al., 2008 ; Souza et al., 2008 ; Bonilla-Rosso et al., 2012 ). Overall, despite its high bacterial diversity, the Archaea domain is underrepresented at several sites in the CCB. Previous metagenomic diversity profiles in two different microbial mats in CCB (red mat and green mat, Bonilla-Rosso et al., 2012 ) showed that bacteria dominated in the red mat, with a relative abundance of 98%, with Pseudomonas as the most abundant genera, along with some representatives of Firmicutes, and Cyanobacteria, while Archaea and Eukarya represented only 1.78 and 0.26%, respectively. Similarly, at the green mat site, a relative abundance of ~93% was found for Bacteria (without a dominant phylum), only 2.06% for Archaea, and 2.79% for Eukaryota ( Bonilla-Rosso et al., 2012 ). Using 16S rRNA gene tags ( Souza et al., 2018 ), 5,167 OTUs (with 97% identity) were detected in soil, sediment, and water samples at different sampling sites in the Churince system in CCB (now a defunct hydrological system). This diversity represented 60 different phyla of microorganisms, of which only three belonged to the Archaea ( Souza et al., 2018 ). However, this changed in March 2016, when an unexpected rain exposed a shallow pond (see Figure 1 ) on the Pozas Azules ranch of Pronatura Noroeste (26° 49′ 41.7” N, 102° 01′ 28.7” W) within the CCB. This pond is characterized by dome-shaped structures that emerge around orange circles; these structures are only observed under humid conditions following a heavy rainfall. Within these elastic dome structures, there is an anoxic, carbon dioxide and methane-rich interior which reminisce what the atmosphere could have been during most of the Archean Eon, before the oxygenation of the atmospheres and oceans, hence the name “Archean domes” (AD) (see Espinosa-Asuar et al., 2022 ; Medina-Chávez et al., 2023 ; Madrigal-Trejo et al., 2023 ). A pH of 9.8 and a salinity of 5.28% were measured during the rainy season, while in the dry season the pH is 5 and the salinity reaches saturation. During the rainy season, a stoichiometric imbalance of C:N:P of 122:42:1 has been reported ( Espinosa-Asuar et al., 2022 ; Medina-Chávez et al., 2023 ; Madrigal-Trejo et al., 2023 ). Figure 1 AD site in Cuatro Ciénegas Basin (CCB). (A) Aerial view of the site. (B) Photo of CCB in 2016 when the site was first explored. The orange circles mark the prominent areas of the site that were investigated. (C) Dome-shaped structures called Archaean Domes. Photo credit: David Jaramillo. An initial study conducted in 2016 on microbial mat diversity at the AD site by Medina-Chávez et al. (2023) revealed that the relative abundance of the Archaea domain reached approximately 5%, encompassing 5 Archaea phyla, 25 orders, 36 families, 93 genera and 230 species, higher than the abundances reported in analyses at other sites in the CCB mentioned above, where the relative abundance of Archaea barely reached 2.0% ( Bonilla-Rosso et al., 2012 ; Souza et al., 2018 ). Subsequent studies at the AD site reported a significant increase in the relative abundance of Archaea, of ~30.60% in 2019, with the phylum Euryarchaeota being the most abundant ( Madrigal-Trejo et al., 2023 ). Given the high level of endemism in CCB and the remarkable diversity of Archaea in the AD site, we anticipate the discovery of numerous new lineages through the use of Metagenome-assembled genomes (MAGs). Therefore, in this study, we aim to deepen the analysis of MAGs found in the AD ponds, both in the dome formation zone and in the adjacent orange circles. Herein we show that MAGs approach provides a more comprehensive overview of the microbial diversity in AD pond than previous studies based on 16S Tags or metagenomics. Through this exhaustive analysis, we obtained a comprehensive set of 325 MAGs from the AD, 48 MAGs belonging to the Archaea domain and 277 MAGs belonging to the Bacteria domain were identified. These genomes represent a broad spectrum of previously unreported microorganisms, encompassing a total of 40 phyla, with 32 belonging to Bacteria and 8 to Archaea.",
"discussion": "4 Discussion 4.1 Taxonomic composition: bacteria prevail over archaea in the AD site The taxonomic analysis of the Archean Domes (AD) site in the Cuatro Ciénegas Basin (CCB) revealed a diverse population of archaea with a relative abundance of 14.45%, especially the phylum Euryarchaeota. This contrasts with other CCB locations, where Archaea typically represent only about 2% of the microbial community ( Souza et al., 2008 ; Bonilla-Rosso et al., 2012 ). This finding highlights the uniqueness of the Archean Domes site, as previously suggested by ( Espinosa-Asuar et al., 2022 ; Medina-Chávez et al., 2023 ; Madrigal-Trejo et al., 2023 ). Euryarchaeota. An alternative to the diversity of archaea could be explained by possible movements of the deep aquifers that brought microorganisms from the deep biosphere to the surface. This could have influenced the microbiological diversity at the AD site and in other ponds within the CCB. The studies by Madrigal-Trejo et al. (2023) , Cisneros-Martínez et al. (2023) and Wolaver et al. (2012) have also observed the possible connection and influence of the movement of deep aquifers. The diversity of archaea at the AD site can be attributed to the salinity conditions present in the environment. The Euryarchaeota is the most predominant, as observed in other places with high salinity ( Fernández et al., 2014 ; Wang et al., 2022 ). However, bacteria are generally more diverse than archaea. Taxa such as Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Cyanobacteria are among the most common. According to Nonpareil curves, our metagenomes have an average coverage of 60 to 70%. However, some samples have even lower coverage, reaching 20% in some cases. This implies that there is still a significant proportion of diversity to explore in our samples. For example, has been reported that the soil samples required greater sequencing effort to achieve almost complete coverage, suggesting their complexity ( Rodriguez-r and Konstantinidis, 2013 ). Nonpareil curves are useful in revealing distinctive characteristics of samples, such as the skewed distribution of species abundance, and tell us when a sufficient proportion of the diversity present in the sample has been sequenced. In our case, some samples barely reach 20% coverage, suggesting much to discover. 4.2 Taxonomic novelty of AD site This is the first time the metagenome-assembled (MAG) genomes were used to expand our understanding of the CCB prokaryotic tree of life, unlike previously published studies that used 16S rRNA tags or metagenome assemblies. Our analysis revealed the presence of several predominant phyla, including Proteobacteria, Cyanobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Spirochaetes, Chloroflexi, and Euryarchaeota, which is consistent with previous studies of diversity at the AD site ( Espinosa-Asuar et al., 2022 ; Madrigal-Trejo et al., 2023). As mentioned in the preceding paragraphs, the AD site harbors microorganisms that render this environment unique. As an example, we obtained the MAG D1M01_16 classified as Marinisomatota phylum, which is a recently proposed bacterial candidate phylum formerly known as SAR406, MGA, or Marine Group A. These bacteria are predominantly found at great depths, such as the Challenger Deep, the Mariana Trench, and the Puerto Rico Trench ( Tarn et al., 2016 ). This phylum exhibits low representation in shallow pelagic samples and high abundance in deep samples. Although these bacteria are often associated with low levels of dissolved oxygen environments, little is known about their ecology and metabolic functions. Marinisomatota is part of the FCB group, alongside other related bacterial phyla. This MAG, along a wealth of data obtained since 2000 ( Souza et al., 2006 , 2012 , 2018 ; Alcaraz et al., 2008 ) supports marine ancestry of CCB despite geological indications that marine waters left the valley with the uplift of the Mexican Sierra Madre Oriental and the closure of the Wester Sea Way 35 MYA. Thus, the CCB has two of the ingredients for hyper-diverse microbial endemism: isolation and long-term continuity. Moreover, these MAGs have enabled us to identify 12 high-quality genomes, as per the MIMAG criteria. Among these, only two MAGs, D1M03_19 and D1M06_10, were classified at the genus level: Puniceicoccus and Wenzhouxiangella , respectively. Moreover, within this set of genomes, the presence of three high-quality genomes from the Archaea domain is noteworthy: D4M11_35 (class Bathyarchaeia), D1M06_39 (order Methanomassiliicoccales), and D4M11_51 (family Bilamarchaeaceae). Given the limited number of reported Archaea genomes in the NCBI database (i.e., only 581 genomes reported as completely sequenced, as of the manuscript writing date), this study makes a substantial contribution to enhancing the taxonomic sampling within the Archaea domain. Moreover, most of the MAGs found in this study represent —at least at the species level—previously unknown taxa. This is not surprising, considering that many of the phyla in which our MAGs are classified have only recently been described. For instance, the Candidate Aenigmarchaeota is an archaeal cluster first identified in 2013 as part of a study of “microbial dark matter” ( Rinke et al., 2013 ). The same is true for the Candidate Woesearchaeota phylum and which was also recently described ( Castelle et al., 2015 ). The diversity within CCB AD site is notable, demonstrated by the distribution of its MAGs across nearly the entire prokaryotic tree of life, spanning both Bacteria and Archaea (refere to Figures 4 – 6 ). In comparison to other hypersaline sites, only Shark Bay (blue hole mats) ( Kindler et al., 2021 ) and Lake Hillier ( Sierra et al., 2022 ) have reported members within the Asgard, TACK, DPANN, and Euryarchaeota superphyla coexisting within the same microbial mat environment. This highlights the unique nature at the AD site and its potential as a source to study microorganisms’ evolution and adaptation in extreme environments. Euryarchaeota, Asgard, and DPANN were also reported in Guerrero Negro ( García-Maldonado et al., 2022 ). Euryarchaeota, TACK, and DPANN presence has been reported in High-Bourne Cay ( Khodadad and Foster, 2012 ). Previous studies have revealed varying compositions of prokaryotic communities across different hypersaline environments. For instance, in Lake Magadi ( Kambura et al., 2016 ) and Hamelin Pool ( Ruvindy et al., 2015 ), reports have primarily focused on Euryarchaeota and TACK members. In contrast, Cape Recife ( Waterworth et al., 2020 ) exhibits a broader diversity, including Asgard and DPANN members. However, some analyses have primarily highlighted the dominant relative abundance of specific taxa within single phyla. For instance, in the hypersaline pool Lake Tyrrell ( Andrade et al., 2015 ), Haloquadratum species and unculturable members of Halobacteriaceae are predominant. Similarly, in the Dead Sea ( Jacob et al., 2017 ), Euryarchaeota and Nanohaloarchaeota dominate. At the Hammam Essalihine site ( Adjeroud et al., 2020 ), the representation of Archaea is relatively weak, primarily comprising members of Parvarchaeota and Crenarchaeota. Meanwhile, in the Salar de Atacama sites (Laguna Brava and Tebenquiche), ( Kurth et al., 2021 ), Lago Diamante ( Rascovan et al., 2015 ), Socompa ( Kurth et al., 2017 ), and Rottnest Island ( Mendes Monteiro et al., 2020 ), only representatives of Euryarchaeota have been reported. 4.3 Understanding the functional landscape of MAGs from AD site The functional annotation of MAGs obtained from AD suggests that certain members of the Archaea play a fundamental role in the carbon cycle. For example, MAG C1M08_23 has the highest level of genes associated with the carbon cycle. Its taxonomic classification according to GTDB-tk corresponds to the Euryarchaeota Phylum, genus Methanohalobium . It is worth mentioning that this genus contains only one species described so far: M. evestigatum ( Zhilina and Merkel, 2019 ), which is halophile and extremely thermophilic, and lives in the hypersaline lagoons of the Arabat spit (East Crimea). It has been reported that this Archaea lives exclusively on the production of methane, either by reducing carbon dioxide with hydrogen or by using methyl compounds as substrates. In the sulfur cycle context, it is evident that Bacteria within the Desulfobacteria phylum play a central role. Out of 325 MAGs, 53 belong to this phylum, distributed across different taxonomic orders: 26 Desulfovibrionales, 20 Desulfobacterales, 4 Desulfatiglandales, 1 Desulfobulbales, 1 Desulfobaccales and a MAG from the class Syntrophobacteria. It has been reported that almost all bacteria from these orders are sulfate-reducing microorganisms, that is, they can perform anaerobic respiration utilizing sulfate as terminal electron acceptor, reducing it to hydrogen sulfide ( Muyzer and Stams, 2008 ; Ward et al., 2021 ). It has also been suggested that they may have contributed to the sulfur cycle shortly after the origin of life on Earth, making them potential ancestors of many microorganisms in a geological context ( Wasmund et al., 2017 ). Regarding the oxygen and nitrogen cycles, cyanobacteria are expected to have an overrepresentation of genes associated with these cycles. This expectation stems from the fact that cyanobacteria are primary producers ( Chen et al., 2022 ). This primary productivity usually occurs through photosynthesis, which uses light as an energy source ( Hamilton et al., 2015 ). However, primary productivity can also occur through chemoautotrophy, which uses the oxidation or reduction of inorganic chemical compounds as an energy source ( Stal, 2012 ). For instance, out of the 13 cyanobacterial MAGs reported in this study, four were classified within the genus Coleofasciculus . Microorganisms of this genus have been reported as one of the most abundant in the microbial mats of the hypersaline lagoon system of Araruama in Brazil (HLSA) ( Walter et al., 2021 ). This suggests that the high abundance of microorganisms of this genus is likely due to their tolerance to high saline levels and their metabolic flexibility (i.e., ability to perform both photosynthesis and anoxic fermentation) ( Burow et al., 2012 ; Walter et al., 2021 ). On the other hand, MAGs D1M13_3 and D1M04_3 classified within the Spirulinaceae family have the highest MEBS index values associated with oxygen cycle genes. These MAGs are phylogenetically close to the cyanobacterial strain ESCF-1, which has been shown to be an important diazotroph in the intertidal microbial mat system in Elkhorn Slough ( Everroad et al., 2016 ), and it has been shown to produce a considerable external carbon pool in the form of EPS (Extracellular polymeric substances). This EPS are managed by an active exoproteome and provides a source of organic carbon for cyanobacteria and other community members ( Stuart et al., 2015 ). Genes related to the iron cycle are also overrepresented in members of Proteobacteria. For example, in MAGs D1M05_10, D1M04_19, and D1M06_10, all of which belong to the genus Wenzhouxiangella , these genes are well represented. Some isolates of this genus were obtained from environments with physicochemical conditions like those described in AD, including alkaline pH and high salt concentration. An example of such isolates is the Wenzhouxiangella strain AB-CW3 ( Sorokin et al., 2020 ), which was obtained from a system of hypersaline alkaline soda lakes in the Kulunda steppe. In this strain, the presence of mtrAB-like genes was reported, which are part of an electron transport system known for iron-reducing bacteria. In this context, these genes may play a role in iron uptake ( Sorokin et al., 2020 ). It is evident that the Archaea belonging to the DPANN superphylum, due to their reduced genomes, do not have an overrepresentation of genes related to any of the C, N, O, S, or Fe cycles. These organisms are characterized by limited metabolic capabilities, with both catabolic and anabolic capacities being significantly limited ( Dombrowski et al., 2019 ). This suggests that at least some members of this superphylum may function as obligate symbionts. The PCA analysis conducted in this study provides crucial insights into the functional diversity of microbial communities within the AD. Dimension 1, explaining 64.9% of the variance, highlights the significant contribution of metabolic pathways like “Pyruvate metabolism,” “Glycolysis/Gluconeogenesis,” and “Methane metabolism” to ecosystem functioning. This underscores the metabolic versatility of microbial communitie inhabiting the AD and their pivotal role in driving biogeochemical processes. Of particular interest is the high loading of “Sulfur metabolism” on Dimension 1, emphasizing the importance of sulfur cycling mediated by microbial communitie in the AD. Dimension 2, explaining 16.3% of the variance, reveals distinct patterns of functional variation, with pathways related to amino acid metabolism and antimicrobial resistance playing key roles in functional differentiation among microbial communities within the AD ( Band and Weiss, 2014 ). The significant loading of “Methane metabolism” underscores the importance of methane as a primary carbon and energy source in AD site. Methanogenic archaea and methane-oxidizing bacteria are central to methane cycling, influencing ecosystem dynamics ( Kharitonov et al., 2021 ). The absence of clear separation between archaeal and bacterial MAGs in terms of their metabolic potential raises intriguing questions about functional redundancy and ecological roles within the AD. This suggests potential functional redundancy within microbial communitie or niche partitioning, warranting further investigation. Regarding the functional annotation of MAG C1M08_23 ( Methanohalobium ), it is important to note that, as mentioned in the results, in the NCBI database, there is only one fully sequenced genome of this genus ( Methanohalobium evestigatum Z-7303) and another at the scaffold level (with assembly identifier GCA_018609725.1). Thus, this work contributes to a better understanding of the metabolism and ecology of the archaeal genus Methanohalobium , which has been reported to be strictly anaerobic and exclusively sustains itself through methane production via the reduction of carbon dioxide with hydrogen or by utilizing methyl compounds as substrates. These species are only moderately halophilic but extremely thermophilic. Based on the results obtained in this study, we can suggest that it is a potentially methanogenic archaea capable of fixing carbon through the Wood-Ljundahl pathway and possibly able to fix nitrogen. This capability, on the other hand, provides a broader perspective in the field of nitrogen fixation. Biochemical and genetic studies demonstrate that nitrogen fixation in Archaea is evolutionarily related to nitrogen fixation in Bacteria and operates through the same fundamental mechanism ( Leigh, 2000 ; Gaby and Buckley, 2014 ). At least three nif genes present in Bacteria (nif H, D, and K) are also found in MAG C1M08_23, suggesting that it may be a diazotrophic methanogenic archaea. Furthermore, this genome suggests the capability for dissimilatory sulfur reduction, as well as dissimilatory sulfur reduction and oxidation. Finally, as shown in Figure 7B , multiple two-component systems were found, which could assist this microorganism in thriving in an extreme environment such as the AD pond. For instance, this MAG encodes for PhoR and SenX3 ( James et al., 2012 ), reported to be involved in the regulation of gene expression under phosphorus-limiting conditions. Methanohalobium also encodes for multiple histidine kinases, such as KinABCDE, which regulate entry into the stationary phase and sporulation, possibly homologous to genes already reported in Bacillus subtilis ( Tojo et al., 2013 ). Additionally, this MAG encodes for NtrY and GlnL, two two-component systems of the NtrC family related to conditions of low nitrogen availability. Regarding the metabolic description of MAG D4M11_7 (Desulfatiglandaceae family), it is suggested that this microorganism has the capability to perform assimilatory sulfur reduction, as well as dissimilatory sulfur reduction and oxidation. Additionally, it incompletely harbors genes associated with the carbon fixation cycle through the Wood-Ljungdahl pathway. However, it is not possible to conclusively state whether this organism is fully capable of executing this pathway, if the genes are present as vestiges, or if the missing genes necessary to complete the pathway are absent due to assembly challenges (87.49% according to CheckM, refer to this value and other values related to MAG quality in Supplementary Table S4 ) or a true lack of the genes in the MAG. Likewise, it is important to note that this MAG encodes for some genes related to methanogenesis, such as the heterodisulfide reductase (HDR). This enzyme, crucial in the Wolfe cycle of methanogenic archaea that generate methane from CO2 and H2, catalyzes the reduction of heterodisulfide (CoM-S–S-CoB) to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Additionally, it encodes for the enzyme tetrahydromethanopterin S-methyltransferase, which catalyzes the transfer of methyl groups from methyl-tetrahydromethanopterin to 2-mercaptoethane-sulfonate and has been identified in the methane-synthesizing complex of Methanobacterium thermoautotrohicum . However, we have not found evidence that this gene encodes for methyl coenzyme M reductase (MCR), which catalyzes the terminal step in biogenic methane production ( Aguinaga Casañas et al., 2015 ). According to the functional annotation of this MAG, it is possible that this microorganism could convert extracellular nitrate to ammonium. The involvement of microorganisms from the order Desulfobacterales in the nitrogen cycle has been previously demonstrated, as the nitrate reduction by Desulfobacterales has been observed to efficiently alleviate nitrogen pollution in the subtropical mangrove ecosystem in the Beibu Gulf in China ( Nie et al., 2021 ). Like the MAG C1M08_23 of Methanohalobium , MAG D4M11_7 encodes for multiple two-component systems such as PhoR and SenX3, also present in MAG C1M08_23, along with multiple histidine kinases such as KinABCDE that regulate entry into stationary phase and sporulation, as well as NtrY and GlnL, which are two two-component systems of the NtrC family related to conditions of low nitrogen availability. 4.4 Monophyletic MAG groups suggest endemicity to CCB The AD site exhibited the presence of the recently described candidate phylum Bipolaricaulota ( Hao et al., 2018 ), which showed monophyletic clustering. Similarly, our phylogenetic analysis of the cyanobacteria group and PVC also showed the formation of such monophyletic groups. These findings suggest that the groups analyzed, as well as other groups showing similar phylogenetic patterns, likely represent endemic groups. The observed clustering pattern could be related to oligotrophic conditions characterized by limited phosphorus availability reported at the site, as evidenced by a reported C:N:P ratio of 122:42:1 ( Espinosa-Asuar et al., 2022 ; Medina-Chávez et al., 2023 ; Madrigal-Trejo et al., 2023). Previous studies have suggested that the low phosphorus (P) and other conditions in CCB have triggered an evolutionary response among its endemic microorganisms ( Souza et al., 2018 ) exemplified by B. coahuilensis . Remarkable adaptations to the environment have been observed in B. coahuilensis , including its ability to produce sulpholipids instead of phospholipids ( Alcaraz et al., 2008 ). This adaptation is attributed to the absence of genes responsible for synthesizing P-rich teichoic acids and polyanionic teichuronic acids ( Souza et al., 2008 ). Furthermore, we conducted a recruitment analysis using 17 genomes reported as reference in the NCBI database (refer to Supplementary Table S4 for more information on these genomes). This analysis revealed minimal genome coverage when compared to the reference genomes, suggesting that these MAGs possess unique genomic characteristics not found in previously reported genomes. These findings align with our observations of the phylogenetic similarity of MAGs from the AD site and their distinctiveness from microorganisms found at other locations, such as the comparison with Halothece sp. PCC 7418."
} | 7,609 |
28424712 | PMC5371606 | pmc | 6,112 | {
"abstract": "Arbuscular mycorrhizal fungi (AMF) are crucial components of fertile soils, able to provide several ecosystem services for crop production. Current economic, social and legislative contexts should drive the so-called “second green revolution” by better exploiting these beneficial microorganisms. Many challenges still need to be overcome to better understand the mycorrhizal symbiosis, among which (i) the biotrophic nature of AMF, constraining their production, while (ii) phosphate acts as a limiting factor for the optimal mycorrhizal inoculum application and effectiveness. Organism fitness and adaptation to the changing environment can be driven by the modulation of mitochondrial respiratory chain, strongly connected to the phosphorus processing. Nevertheless, the role of the respiratory function in mycorrhiza remains largely unexplored. We hypothesized that the two mitochondrial respiratory chain components, alternative oxidase (AOX) and cytochrome oxidase (COX), are involved in specific mycorrhizal behavior. For this, a complex approach was developed. At the pre-symbiotic phase (axenic conditions), we studied phenotypic responses of Rhizoglomus irregulare spores with two AOX and COX inhibitors [respectively, salicylhydroxamic acid (SHAM) and potassium cyanide (KCN)] and two growth regulators (abscisic acid – ABA and gibberellic acid – Ga3). At the symbiotic phase, we analyzed phenotypic and transcriptomic (genes involved in respiration, transport, and fermentation) responses in Solanum tuberosum/Rhizoglomus irregulare biosystem (glasshouse conditions): we monitored the effects driven by ABA, and explored the modulations induced by SHAM and KCN under five phosphorus concentrations. KCN and SHAM inhibited in vitro spore germination while ABA and Ga3 induced differential spore germination and hyphal patterns. ABA promoted mycorrhizal colonization, strong arbuscule intensity and positive mycorrhizal growth dependency (MGD). In ABA treated plants, R. irregulare induced down-regulation of StAOX gene isoforms and up-regulation of genes involved in plant COX pathway. In all phosphorus (P) concentrations, blocking AOX or COX induced opposite mycorrhizal patterns in planta : KCN induced higher Arum -type arbuscule density, positive MGD but lower root colonization compared to SHAM, which favored Paris -type formation and negative MGD. Following our results and current state-of-the-art knowledge, we discuss metabolic functions linked to respiration that may occur within mycorrhizal behavior. We highlight potential connections between AOX pathways and fermentation, and we propose new research and mycorrhizal application perspectives.",
"introduction": "Introduction The political, social and economic context should, in the next years, favor the exploration and exploitation of beneficial soil organisms for crop production. Government policy initiatives (European Directive 2009/128/EC) and consumer demand will lead to alternative production methods in order to reduce the use of phytosanitary products and fertilizer inputs. Thus, agroecological strategies are increasingly explored and recall some basic definitions that (re)integrate the plant in its environment. The soil is the first habitat for plants which continues to interact at all stages of plant’s life cycles ( Jeffries et al., 2003 ). The soil is also one of the main reservoirs of ecosystem services found on Earth, provided by a wide range of microorganisms, including bacteria and some soil fungi, such as arbuscular mycorrhizal fungi (AMF; Gianinazzi et al., 2010 ). The predominant mutualistic symbiotic relationship between AMF and plant roots was established over 400 million years ago ( Brundrett, 2002 ) in more than 200,000 species, which belong to 74% of plant families ( van der Heijden et al., 2015 ). AMF are obligate biotrophs, represented by at least 289 species 1 found worldwide under a wide range of ecological conditions. Many positive mycorrhizal effects on host plants have been reported. AMF can (i) improve plant growth by a better transfer of water and inorganic nutrients, especially phosphorus ( Smith and Read, 2008 ); (ii) increase plant-pathogen resistance and plant health ( Whipps, 2004 ; Pozo et al., 2009 ); (iii) boost plant photosynthesis ( Quarles, 1999 ); (iv) stabilize soil by the excretion of the fungal glycoprotein, glomalin ( Rillig and Steinberg, 2002 ; Bedini et al., 2009 ); (v) alleviate the impact of abiotic stresses such as cold or heat ( Volkmar and Woodbury, 1989 ; Charest et al., 1993 ; Zhu et al., 2010 , 2011 ), salinity ( Porras-Soriano et al., 2009 ), drought ( Aroca et al., 2007 ), nutritive starvation ( Smith and Read, 2008 ) or heavy metals ( Karimi et al., 2011 ). In return, it is believed that AMF benefit from the plant’s carbohydrates supply ( Bago et al., 2003 ) associated with the stimulation of fatty acid synthesis in fungal hyphae ( Trépanier et al., 2005 ). Arbuscular mycorrhizal fungi are probably one of the essential components of the “second green revolution” ( Lynch, 2007 ), but their implementation faces some major difficulties, namely restrictions from plant producer’s perspective, product costs, producer awareness level and variability in mycorrhizal inoculum quality ( Vosátka et al., 2008 ; Ijdo et al., 2011 ; Berruti et al., 2016 ). But there are also limitations inherent to the biological system itself since mycorrhizal benefits are not always guaranteed ( Vosátka et al., 2008 ; Ijdo et al., 2011 ; Malusá et al., 2012 ) and the physicochemical properties of targeted soils can negatively impact the symbiosis ( Smith and Smith, 2011a , b ). One of the biggest challenges of mycorrhizal inoculum field application is the high phosphorus (P) content often encountered under conventional cropping, due to P fertilizer input. It is known that high phosphorus concentrations (or its inorganic salt phosphate) systematically inhibit mycorrhizal colonization ( Smith and Read, 2008 ; Breuillin et al., 2010 ), and the physiological signaling generated by this element appears systemic since foliar application can lead to the same effects ( Sanders, 1975 ; Schreiner and Linderman, 2005 ; Schreiner, 2010 ). Phosphate affects not only the establishment but also the functioning of mycorrhizal symbiosis. Fungal structure development of the internal mycelium can be divided in two general anatomical groups described by Gallaud (1905) . The Arum -type consists of characteristic highly branched arbuscules within cortical cells, formed from a short side hyphal branch. The Paris -type is characterized by the development of extensive intracellular coiled hyphae, which spreads from cell to cell sometimes with only low rates of arbuscule-like branch formation. McArthur and Knowles (1992) have shown in potato root that AMF develop preferentially Arum -type under low P while Paris -type occurs under high P. These two mycorrhizal types may act in different ways within plant roots. In rice, the symbiotic phosphate transporter (PT) OsPt11 is preferentially active in arbuscule branches but not around coiled hyphae ( Kobae and Hata, 2010 ). Given that increasing phosphorus concentration is often associated with a decrease of mycorrhizal growth response (MGD; Smith and Smith, 2011a ), these data suggest differential plant fitness related to the mycorrhizal type they harbor. Despite numerous studies, there is not yet a complete explanation for the P inhibition. Therefore, there is an urgent need to better understand the physiological bases of this phenomenon in order to define innovative strategies to improve mycorrhizal development and performance, which are sine qua non conditions to realize the mycorrhizal implementation under high P crop field conditions. One obvious connection between P and organism behavior is the mitochondrial respiration activity, in which P plays a crucial role as energetic component of ATP. In most plants and fungi, the respiration yield is modulated by the electron partitioning flow shared between the cytochrome oxidase (COX) and the alternative oxidase (AOX) pathways that take part in the electron transport chain ( Vanlerberghe, 2013 ). Both transfer electrons to O 2 (which results in water formation), but it is usually assumed that AOX is a non-conserving energy pathway because it does not contribute to ATP formation ( Vanlerberghe, 2013 ) and is regulated by the mitochondrial redox status and the glycolytic flux. In plants, the COX pathway involves cytochrome c reductase, cytochrome c and cytochrome c oxidase enzymes. Whereas cytochrome c (Cytc) is composed of a single small polypeptide, cytochrome c oxidase is a multimeric complex composed of several different subunits, encoded by the mitochondrial and the nuclear genome ( Welchen et al., 2002 ). Subunit Vb ( COXVb ) is the most conserved among nuclear-encoded subunits ( Rizzuto et al., 1991 ). Cytc is essential for plant growth and survival and the knock-out of both Cytc genes in Arabidopsis is lethal to the plants while they participate for complex IV stability ( Welchen et al., 2012 ). AOX plays an important role during various stress responses (such as P limitation, Sieger et al., 2005 ; Plaxton and Tran, 2011 ) and in specific developmental phases, depending on the expressed isoform ( Umbach et al., 2006 ; Zsigmond et al., 2008 ; Vanlerberghe, 2013 ). However, its metabolic significance is much less clear but specific metabolic functions must be involved when the AOX pathway is engaged to sustain basal general metabolic processes associated with the a specific redox status (NAD(P) + /NAD(P)H) cell pool in order to cope with energy demand. In this regard, fermentation metabolism activity could play an important role ( Sakano, 2001 ). The best-known function of fermentative metabolism is to recycle NADH to NAD + to avoid the depletion of the cytosolic NAD + pool and inhibition of glycolysis when oxidative phosphorylation is impaired ( Sakano, 2001 ). However, no data is available about the importance of these processes in mycorrhizal symbiosis. In fungi, AOX plays a role in growth regulation and development, resistance, pathogenesis and pathogenicity, and may contribute to fungal ecological fitness ( Umbach and Siedow, 2000 ; Uribe and Khachatourians, 2008 ; Ruiz et al., 2011 ; Grahl et al., 2012 ; Thomazella et al., 2012 ; Xu T. et al., 2012 ). Unlike plants, in which AOX form small multigenic families, the analysis of the fungal genomes currently available reveals that a majority of fungal species possessing the AOX pathway have only one gene sequence, with a maximum of three sequences per genome ( Mercy et al., 2015 ). In particular, very few studies were conducted to elucidate the role of the two electron pathways in AMF, despite their known importance for the growth of many organisms: - It was shown that the COX1 protein content is increased in hyphae ( Besserer et al., 2006 ) while the transcript level of COXIV is increased in hyphae as compared to spores ( Besserer et al., 2008 ) within days succeeding application of strigolactone analogous (GR24) in Gigaspora rosea . Hyphal development seems, therefore, associated with the COX pathway, and it corresponds to a higher NAD(P)H protein activity (concomitant with an increase in NADH dehydrogenase activity) and ATP production observed at hyphal tip ( Besserer et al., 2008 ). - Existence of the cyanide-insensitive respiration pathway in AMF was highlighted by the presence of an AOX sequence in Rhizoglomus irregulare genome, close to the Mucoromycotina AOX 1 ( Campos et al., 2015 ; Mercy et al., 2015 ), but limited functional data were published. By using SHAM as AOX pathway inhibitor, Besserer et al. (2009) suggested a role of AOX during Gigaspora rosea spore germination. Mitochondrial changes (density and respiration) were observed in response to branching factors ( Tamasloukht et al., 2003 ; Besserer et al., 2006 ), which may suggest a role of the AOX or COX pathways during the pre-symbiotic phase. Campos et al. (2015) observed a coincident of up-regulation of the tomato AOX1 genes and down-regulation of the RiAOX gene during the first six weeks of symbiosis establishment. Expression data obtained under cold-stress conditions showed that the presence of AMF is able to induce an opposite plant mitochondrial respiratory pattern, by potentially reversing the electron route pathway from the AOX to the COX ( Liu et al., 2015 ). Note that the growth regulator abscisic acid (ABA) was shown to play a crucial role in arbuscule formation and functionality ( Herrera-Medina et al., 2007 ; Martin-Rodriguez et al., 2010 , 2011 ; Aroca et al., 2013 ), while it regulates the AOX gene expression and activity in plants ( Finkelstein et al., 1998 ; Choi et al., 2000 ; Rook et al., 2006 ; Giraud et al., 2009 ; Lynch et al., 2012 ; Wind et al., 2012 ). Nevertheless, no roles were clearly defined for the respiratory pathways during spore dormancy or the symbiotic phase. Although several studies suggest a connection between respiration and P nutrition, little is known about the uptake and transport of P in connection with the respiratory pathways involved. Plant Pi uptake across the plasma membrane is mediated by Pi/H + symporters belonging to the Pht1 gene family ( Bucher, 2007 ). In mycorrhizal plants, two P uptake pathways were identified: the “direct phosphate uptake” pathway (DPU), mediated by high affinity transporters that are strongly expressed in roots ( Smith et al., 2011 ) and the “mycorrhizal phosphate uptake” pathway (MPU), relying on AM-inducible Pi transporters, crucial for Pi flux across the periarbuscular membrane at the mycorrhizal interface ( Javot et al., 2007 ; Yang et al., 2012 ). Inorganic phosphate transporters are also present on the inner mitochondrial membrane and are represented by two families: the phosphate/dicarboxylate carrier (DIC) and the phosphate carrier Pht3 (here named ‘MPT’ for mitochondrial phosphate transporter). MPTs deliver most of the Pi required by the mitochondrial ATP synthase complex ( Kiiskinen et al., 1997 ). This work is an exploration of the functional framework of the cyanide-sensitive and cyanide-insensitive respiration pathways in the mycorrhizal system Solanum tuberosum / Rhizoglomus irregulare (whose genomes are available). To study this complex aspect in a holobiont system, three strategies were developed: the first assay was implemented to study the impact of respiratory inhibitors SHAM (AOX inhibitor) and KCN (COX inhibitor), as well as, two antagonistic phytohormones (ABA and Ga3) on mycorrhizal spore behavior at the pre-symbiotic phase (axenic condition). The second trial was designed to analyze transcript variations of several genes involved in respiration and fermentation pathways using ABA, a phytohormone known to promote the mycorrhizal symbiosis and also known to be one regulator of the AOX pathway. Then, a third assay consisted to set a non-lethal pharmacological approach using KCN and SHAM treatments, under five different phosphorus concentrations. Our data reveal differential mechanisms that shape plant and fungal behavior by affecting yield, plant FW, mycorrhizal type, hyphal development and MGD. We show that the electron flow partitioning is a key determinant in mycorrhizal behavior and mycorrhizal effects, at least in the S. tuberosum / R. irregulare biosystem. We discuss its potential relevance in regard to specific metabolic pathways, notably to fermentation, but also for the mycorrhizal application.",
"discussion": "Discussion Mitochondrial Respiratory Inhibitors Influence Fungal Behavior at Pre-symbiotic Phase Dormancy is often related to AOX pathway in plant seeds, but also in fungi. Several examples have been reported, especially within Mucoromycotina ( Cano-Canchola et al., 1988 ; Salcedo-Hernandez et al., 1994 ) which are phylogenetically closely related to AMF ( Hibbett et al., 2007 ), showing a respiratory shift characterizing spore germination process from AOX (dormancy) to COX (hyphal growth). It is difficult to define AOX/COX interplay during spore germination in R. irregulare as spores can return into dormancy and germinate again several times without obvious phenotypic signs ( Giovanetti et al., 2010 ), and the hyphal tube germination is the physiological consequence of an already implemented respiratory shift, as shown for Mucor rouxii ( Cano-Canchola et al., 1988 ). Nevertheless, our data (Experiment 1) with KCN suggest that AOX is very likely involved in spore dormancy. Application of SHAM or KCN inhibited the spore germination, supporting the importance of both electron pathways and a possible involvement of a respiratory shift from AOX to COX. However, further work is needed to confirm this statement. AMF spores are sensitive to plant hormones, and their germination responses harbor opposite patterns between the antagonistic hormones ABA and Ga3, as ABA maintained spore dormancy while Ga3 broke it, similarly to plant seeds ( White and Rivin, 2000 ; Linkies and Leubner-Metzger, 2012 ). Therefore, these observations support the need to apply ABA as pre-treatment (Experiment 2) and the need to apply the respiratory inhibitors some days after plant inoculation (Experiment 3) in order to not disturb the pre-symbiotic developmental phase of R. irregulare . Promotion of hyphal branching pattern in ABA treatment fits with previous observations: Juge et al. (2002 , 2009 ) showed that spores develop g-type pattern germination (fine branching hyphae) around spores when dormancy is incompletely broken or under stress conditions, while G-type pattern (runner hyphal growth pattern) occurs in favorable conditions. Hyphal branching from germ tube generated by ABA seems similar to its effect on the arbuscule formation ( Herrera-Medina et al., 2007 ). As a remark, INT staining data suggested a low spore viability in Ga3 and SHAM treatment and higher after ABA and KCN (5 mM) treatment. However, no formazan production was observed in germinated spores following Ga3 application, while the germinative hypha was still growing (data not shown). This suggests that INT staining could correspond to a reducing power marker in the cell, and might be associated to AOX metabolism and spore dormancy, rather than solely to a vital staining. Mitochondrial Respiratory Inhibitors Influence Fungal Behavior at Symbiotic Phase Occurrence of the Arum - and Paris -types are not well understood and corresponds to extremes, which can coexist in a developmental continuum within the same root structure ( Smith and Read, 2008 ). Their formation depends partly on genotypic factors characterizing both partners ( Karandashov et al., 2004 ), partly on environmental factors like phosphate availability ( Smith and Read, 2008 ). In potato roots, both mycorrhizal types and root colonization are commonly influenced by phosphate concentration ( McArthur and Knowles, 1992 ). This is in agreement with our observations since P concentration inhibited mycorrhizal development in a dose-dependent way, and Arum- type structures were occurring at low to medium P concentration (1, 10, and 50 ppm), while Paris -type formation appeared at higher P levels (100 and 300 ppm). Our data on mycorrhized potato suggest that AOX or AOX-related metabolism is involved in arbuscule/hyphal branching and Arum -type formation, while COX or COX-related metabolism is associated with higher hyphal growth and hyphal-coiled shape ( Paris -type) formation. Despite the use of SHAM being controversial because of its possible non-specificity to AOX ( Bingham and Stevenson, 1995 ; Day et al., 1996 ), our results showed differential and opposite phenotypic patterns in plant and fungal behavior when SHAM and KCN treatments were compared. KCN and ABA are known to stimulate AOX activity ( Finkelstein et al., 1998 ; Choi et al., 2000 ; Rook et al., 2006 ; Giraud et al., 2009 ; Millar et al., 2011 ; Lynch et al., 2012 ; Wind et al., 2012 ). These two molecules promoted both arbuscule intensity and branching, but they caused differential mycorrhizal development (M %), with ABA treated plants yielding a higher M % (with colonization in the primary adventitious root recognized under the experimental conditions even up to the base stem). This could be explained by the fact that COX capacity is not inhibited by ABA unlike KCN. On the other hand, we observed that ABA (Experiment 2) tended to promote the AOX gene transcript levels in non-inoculated potato plantlets, but such response was not clearly identified when using KCN (Experiment 3). It would therefore suggest that the mycorrhizal root colonization, but also transcript regulation of the AOX pathway, are dependent from the functional state on the COX pathway. To summarize, we can deduce that mycorrhizal behavior seems to be linked to the mitochondrial respiratory chain-partitioning environment, probably generated from both partners. As a remark, ethylene is another stress hormone that is able to induce AOX pathway ( Simons et al., 1999 ; Wang et al., 2010 ; Xu F. et al., 2012 ), but usually impairs mycorrhizal colonization (using epinastic plant or exogenous application, Zsögön et al., 2008 ; Fracetto et al., 2013 ). This phenomenon could be partly explained by the action of cyanide (HCN) produced stoichiometrically (1:1) with ethylene, blocking therefore the COX pathway ( Xu F. et al., 2012 ). Interpretation of the sporulation rise at 50 ppm P in M plants is challenging, but it seems linked with specific plant metabolism independent of mycorrhizal colonization (M %), as already observed by past studies ( Douds and Schenck, 1990 ), while this last parameter harbored high correlation with P concentration. A particularity is observed at 50 ppm P concentration through the various plant phenotypical parameters studied (plant growth parameters – Supplementary Figure S2 ; mycorrhizal dependency – Figure 8 ) and corresponds to the highest value of arbuscule intensity (a %, Supplementary Figure S1 ) with a predominant Arum -type, concomitant with an up-regulation of RiAOX transcripts ( Figure 9 ) and the highest sporulation ( Figure 5 ). Taking into account all these observations, we propose a scheme defining the roles of AOX and COX in the different mycorrhizal phenotypical behaviors ( Figure 11 ). FIGURE 11 Scheme describing potential roles of AOX and COX metabolism in arbuscular mycorrhizal fungi. This scheme was drawn according to the fungal phenotype observed with SHAM and KCN treatments under in vitro and ex vitro assays. Compared to untreated plants and KCN treatment, SHAM decreases arbuscule intensity within mycorrhizal root fragment (a %), with predominance of Paris –type development, while vesicles intensity remains similar to non-treated plants. Compared to SHAM, KCN plants harbored reduced vesicle within root fragment (v %) and hyphal density (H %), but higher arbuscule formation in mycorrhizal root fragments (a %). Note that the inhibitors response on fungal parameters may differ according to specific P concentration. Spore germination is inhibited by ABA, SHAM, and KCN but enhanced by Ga3. We propose that spore germination corresponds likely to a punctual event involving respiratory shift that transfers a dormant state (likely linked with AOX pathway) to an active state (likely linked with COX pathway) allowing the fungus to explore and reach a host plant root (see text in section, Mitochondrial Respiratory Inhibitors Influence Fungal Behavior at Pre-symbiotic Phase). However, the respiratory chain pathway involved in spore formation and size, hyphal diameter, as well as the filling of storage organs and active cytoplasmic flow in AMF remains unknown. We suggest that COX metabolism might be preferentially expressed in these different processes that require energy (suggestions are written in purple). Mitochondrial Respiratory Inhibitors Induced Opposite Mycorrhizal Growth Dependency Although many factors can influence the MGD, it is usually recognized that high P concentrations decrease MGD, which may become negative ( Smith and Smith, 2011a ). In our study, no correlation was found between MGD and P concentration, but respiratory inhibitors induced opposite responses. All possible MGD responses (neutral for non-treated plants, positive under KCN and negative under SHAM) were observed within the same AMF-plant biosystem, illustrating the high plasticity of mycorrhizal behavior linked to the physiological context. We show the possibility to obtain increasing yield with increasing P concentration when respiration in the plant–AMF system is associated with AOX pathway (i.e., in KCN treatments), occurring with a low fungal colonization and associated with relatively stable and almost maximal positive MGD. This goes together with a predominant Arum -type structures and higher arbuscule intensity when compared to treatments where COX pathway would be engaged (i.e., in SHAM treatments), in which Paris -type structure is predominant and associated with a negative MGD. This is consistent with the study of van Aarle et al. (2005) , showing that Arum -type structures had higher metabolic activity than Paris -type ones. Moreover, even if SHAM reduced the MGD and the arbuscule intensity, while favoring Paris -type structures, the AMF produced a denser mycelium (h %) and as many vesicles as in non-treated mycorrhizal plants (except at 1 ppm P). AMF responses on plant performance seem therefore to depend mainly on the ability to form hyphal branching ( Arum -type), possibly by an increased exchange surface for providing nutrients to plants ( Kobae and Hata, 2010 ; Bapaume and Reinhardt, 2012 ). However, if MGD responses seem related with arbuscule type in KCN and SHAM treatments, no direct relationship was found between arbuscule intensity and MGD. This leads to the hypothesis that although various membrane transporters (phosphate, amino acid, sugar or nitrogen) were characterized in arbuscules, these fungal structures might not constitute preferential sites for nutrient/element exchanges, and a role should be also attributed to intraradical hypha. This assumption is supported by the fact that glucose transporters were observed not only in arbuscules but also on intraradical hyphae ( Helber et al., 2011 ). The mycorrhizal dependency variation has been attributed to plant species and cultivars, fungal species and isolates, host and symbiont interplay, mycorrhizal rate, soil phosphorus concentration and environmental conditions ( Plenchette et al., 1983 ; Singh, 2001 ; Smith and Smith, 2011a ). Our data suggest that the electron flow partitioning, which is modulated by environmental stimuli and genetic background, corresponds to a main metabolic component determining the MGD related to the mycorrhizal type. Finally, AOX metabolism, linked usually with higher ABA content, is known to be related to plant growth regulation and has been reported to be connected to growth depression ( Sieger et al., 2005 ). This may partly explain the negative MGD phenomenon that AMF might trigger during early developmental stages in some plant species ( Koide, 1985 ; Graham and Abbott, 2000 ; Smith et al., 2009 ; Ronsheim, 2012 ) during the implementation of the induced systemic response in the plant ( Pozo et al., 2002 ; Hause and Fester, 2005 ; Hause et al., 2007 ). Mitochondrial Respiratory Inhibitors Influence Symbiotic Nutrient Transport Arbuscular mycorrhizal inducible Pi transporters of the Pht1 family have been described as markers of mycorrhizal development and, in some cases, proposed as markers of mycorrhizal functionality ( Javot et al., 2007 ). Partly in accordance to this statement, our data show a correlation between AM-inducible transporter expression ( StPT3, StPT4 , and StPT5 ) and the mycorrhizal development. A gradual repression of the MPU pathway with increasing P availability in non-treated mycorrhizal plants was also observed, which is in accordance with several previous studies on Solanaceae species ( Chen et al., 2007 ; Nagy et al., 2009 ), suggesting that the MPU is not functional at high P levels. However, the repression of the MPU by P disappeared when plants were treated with KCN, and surprisingly all AM-inducible P transporters were strongly up-regulated at 300 ppm in non-inoculated plants. This last observation deserves further investigations, as no previous works have been done with very high P concentrations to our knowledge. The mycorrhizal impact on DPU is less clear. A number of studies have shown that AM colonization of plants down-regulates the expression of the DPU Pi transporters ( Rausch et al., 2001 ; Burleigh et al., 2002 ; Glassop et al., 2005 ; Requena, 2005 ; Nagy et al., 2006 ), while other studies pointed contrasted transcriptional regulations. Chen et al. (2007) showed a mycorrhiza-induced down-regulation of Pht1;1 and Pht1;2 expression under low-P conditions, but up-regulation of Pht1;2 under high-P conditions for pepper, eggplant and tobacco. In tomato, Nagy et al. (2009) did not find any change in the transcript abundance for SlPT1 and SlPT2 upon root colonization. Our data are in accordance with these latter observations, as presence of AMF did not necessarily induced down-regulation of non-AM-inducible transporters, but responses were specific to P concentrations. For example, it was noticed that AMF induced an up-regulation at 100 ppm P but a down regulation at 10 ppm P for StPT1 , while an up-regulation of StPT2/6 was observed at 100 ppm P. Only StPT2/6 was regulated by P availability. KCN treatment induced an up-regulation of DPU transporters in mycorrhizal conditions. Fungal Pht1 genes are known to be expressed in the intraradical phase ( Harrison and van Buuren, 1995 ; Benedetto et al., 2005 ; Balestrini et al., 2007 ; Tisserant et al., 2012 ; Fiorilli et al., 2013 ; Walder et al., 2016 ). Fiorilli et al. (2013) showed a slight down-regulation of RiPT1 when exposed to higher P concentrations, and Walder et al. (2016) reported a positive correlation of RiPT5 transcript levels and Pi acquisition in Sorghum. In our study, only RiPT5 was slightly regulated by P levels, with up-regulation in concentrations higher than 50 ppm in non-treated conditions. Few data exist on the translocation of Pi across the inner mitochondrial membrane by Pht3 family in plants and fungi. In our study, no clear role emerged for these transporters within AMF symbiosis. StMPT3 , which presented the highest expression levels in the roots, appeared to be constitutive, and only StMPT1a was slightly up-regulated by the presence of R. irregulare in non-treated conditions. RiMPT appeared also to be constitutively expressed in our experimental conditions. In plants, several transcriptomic analyses revealed that AM establishment can induce the expression of plant N transporters, mainly in arbusculated cells ( Gomez et al., 2009 ; Guether et al., 2009 ; Kobae et al., 2010 ; Gaude et al., 2012 ; Ruzicka et al., 2012 ). AMTs identified in tomato LeAMT4 and LeAMT5 were reported to be exclusively expressed in mycorrhizal roots and not regulated by NH 4 + ( Ruzicka et al., 2012 ). The two potato homologs, StAMT4 and StAMT5 , were strongly up-regulated by AMF, and were gradually repressed by P in mycorrhizal plants. This decrease in transcript levels can be attributed to the reduction of fungal structures inside the roots. However, in the case of AM-inducible AMTs, such as AM-inducible Pht1, NM plants presented a strong up-regulation at a high P level (300 ppm). As for P transporters, a suppression of this up-regulation at 300 ppm P in the presence of KCN was observed and could be explained by the need of a functional COX pathway for ammonium and phosphate transport. NH 4 + uptake via AMT seems indeed to be accompanied by H + extrusion by the plasma membrane H + -ATPases for maintenance of the cytosolic charge balance, and increased fluxes of NH 4 + would increase the demand for respiratory ATP ( Britto and Kronzucker, 2005 ). Hachiya et al. (2010) suggested that the ammonium-dependent increase of the O 2 uptake rate can be explained by the up-regulation of the cytochrome pathway, which may be related to the ATP consumption by the plasma-membrane H + -ATPases. Similarly, P transport is a proton-dependent process, and the H + -ATPase HA1 of Medicago truncatula was shown as essential for P transport in AM symbiosis ( Krajinski et al., 2014 ). StHA1 is also up-regulated at 300 ppm P in non-inoculated plants compared to 1 ppm P and repressed in mycorrhizal plants. It can be therefore hypothesized that an impaired COX pathway would repress ammonium transport via AMT gene family members. P and N AM-inducible transporters were found to be connected to the mycorrhizal development in non-treated plants. On the other hand, no correlation was observed between the expression of P and N AM-inducible transporters and the presence of either specific fungal structures (arbuscule or intraradical hypha) or any MGD parameters across the treatments (Supplementary Tables S2–S4 ). In particular, M KCN plants were associated with positive MGD compared to NM KCN plants but the transcript levels of genes encoding AM-inducible transporters were similar to M SHAM treatments. Several studies showed a lack of relationship between Pht1 gene expression and mycorrhizal Pi acquisition ( Grace et al., 2009 ; Grønlund et al., 2013 ; Walder et al., 2015 ). It would therefore indicate that MGD and that plant’s Pi acquisition through the MPU is not quantitatively regulated by the expression level of AM-inducible Pht1 genes. These observations, combined with the lack of repression of the MPU by P in presence of KCN, and the strong up-regulation of AM-inducible P transporter genes at 300 ppm in NMs, suggest that these transporters are not suitable markers for a functional symbiosis in field conditions where high P concentrations can occur and natural or anthropogenic source of cyanides can be found in soil or water (with concentrations as high as 100 mg kg DW -1 ). What about the Metabolic Role of AOX and COX in AMF? Despite sugar transporters being found and characterized ( Helber et al., 2011 ), AMF are still unable to complete their life cycle even when carbon sources are added in in vitro culture systems without the presence of plant roots. Some publications showed even the opposite: glucose and fructose application under axenic or monoxenic conditions resulted in reduced fungal growth or spore germination rate ( Mosse, 1959 ; Koske, 1981 ; Hepper, 1982 ; Siquiera and Hubbell, 1986 ; Wang et al., 2015 ), while culture media devoid of sucrose can stimulate spore germination, hyphal growth ( D’Souza et al., 2013 ), as well as, sporulation ( St-Arnaud et al., 1996 ). In many publications, the definition of obligate biotrophy of AMF deals with their dependency on plants for carbohydrate supply (mainly glucose and fructose, Pfeffer et al., 1999 ). But obviously, this statement seems incomplete since it is not yet strictly proven by the successful implementation of an axenic culture. Genomic and transcriptomic data obtained from R. irregulare were not helpful since this fungal species would possess all the necessary genes to harbor saprobe behavior ( Tisserant et al., 2012 ; Tisserant et al., 2013 ), although no evidence for gene encoding for fungal multi-domain fatty acid synthase was found ( Wewer et al., 2014 ). Therefore, the fungal needs related to the biotrophy of AMF remains still an open question. In this way, it seems important to emphasize that plants provide, at least, a habitat, i.e., a physical growth support associated with a favorable physiological frame allowing uptake and metabolic assimilation of carbon sources that AMF seems not to encounter without host. This questions the definition of the metabolic frame (induced by stress signals) favorable to AMF colonization, arbuscule development and functions on plant performances. We discuss below the role of COX and AOX, which can shed new light that would allow a better understanding and mastering of mycorrhiza. COX Pathway and Aerobic Respiration In non-inoculated plants, fresh biomass and potato yield are repressed by KCN and enhanced by SHAM (especially at 100 and 300 ppm P). This suggests that the tuberization is an active COX-dependent process needing O 2 , which is consistent with the high O 2 concentration requirement observed previously for tuber formation ( Saini, 1976 ; Hooker, 1981 ; Cary, 1985 ; Geigenberger et al., 2000 ). This fits also with the reported effect of SHAM, known to optimize the O 2 flow through plant tissues ( Sesay et al., 1986 ; Spreen Brouwer et al., 1986 ; Møller et al., 1988 ; Gupta et al., 2009 ). The positive MGD on yield observed in KCN treatment suggests that AMF improve plant respiration, not only related to P concentration, but also to tissue oxygenation, in order to sustain both COX needs and a better carbon allocation in the tuber. However, our expression data obtained at harvest for StCOXVb and StCytc1 genes are difficult to interpret in link with these assertions. StCytc1 expression is up-regulated in NM KCN plants compared to NM SHAM plants at 10 and 100 ppm P, but it remains similar for the other P concentrations tested, suggesting probable other regulation levels related to COX pathway. Potassium cyanide impacted negatively intraradical hyphal growth and vesicle formation, likely by blocking O 2 consumption needed for fungal COX metabolism. It is known that anaerobic conditions in flooded soils is associated with reduced mycorrhizal colonization ( Miller and Sharitz, 2000 ). Moreover, arbuscules are resistant to KCN when compared to non-treated plants, but their intensity is promoted compared to SHAM in all P concentrations, increasing, therefore, the arbuscule/intraradical hypha ratio. This is consistent with the results of Saif (1981) , where soil O 2 content was correlated with the mycorrhizal development and vesicles production, and arbuscule occurrence was observed only at specific O 2 concentration but not under soil O 2 excess. Depression of arbuscule intensity by SHAM might be related to a higher O 2 availability for AMF respiration in roots. The sensitivity of arbuscules to O 2 raises the possibility that they might be a respiration structure where gas exchanges between both partners might occur. Soils have usually less available O 2 ( Clark, 1967 ; Bhattarai et al., 2005 ) and are enriched in CO 2 compared to ambient atmosphere ( Bouma and Bryla, 2000 ; Pfanz et al., 2004 ). As Miller and Bever (1999) suggested, O 2 could be provided by the plant and mobilized for COX-metabolism needs for fungal growth. The increase of mycorrhizal development in ABA treated plants could be linked to a promoted plant COX pathway potential, this hypothesis being supported by the induction of StCytc1 gene in M+ABA treatment. AOX Pathway and Fermentation Our data showed that AOX is involved in specific plant and fungal behavior. However, its energetic metabolic significance is not yet well understood. In order to better interpret its functions within mycorrhizal symbiosis, we thought it was relevant to enlighten possible connections with fermentation, as one “AOX-related key metabolism.” Fermentation pathways use pyruvate as source that can be reduced to lactate (by LDH) in presence of reducing potential (NADH) or processed into CO 2 and acetaldehyde (by PDC, a thiamine pyrophosphate-dependent enzyme), which is then reduced into ethanol (alcohol dehydrogenase) or acetate (aldehyde dehydrogenase), also in presence of NADH. Pyruvate can be synthesized from glucose (classic glycolytic pathway) or from malate (alternative glycolytic pathway) depending on the pH-stat and therefore redox potential ( Sakano, 2001 ). Several clues from previous metabolic studies suggest a possible link between AOX and fermentation in plants but also in fungi and are detailed in Supplementary Material (Section 3). In our study, P concentration was not directly correlated with fermentation in non-treated conditions. But we noticed in Experiment 3 an induction of StLDH1 and StPDC3 occurring at several P concentrations, when comparing KCN to SHAM treatments. Moreover, our data from the ABA test showed that expression of genes involved in fermentation in non-inoculated plants were associated with the higher expression of the genes encoding for AOX isoforms and lower StCytc1 . These observations would indicate persistent implementation of the fermentative system under stressed conditions, which was expected in these situations. From our data, the sporulation pattern in non-treated plants was not correlated with mycorrhizal colonization, while this last parameter harbored high correlation with P concentration. This could suggest that spore formation and mycorrhizal development, requiring obviously energy for their development, may need the involvement of at least two carbon source partitioning. We noticed a significant correlation between sporulation and the StLDH1 profile, both showing, with the RiAOX transcripts, maximum values at 50 ppm P (non-treated plants). At the same time, the presence of AMF reduced significantly the total biomass only at 50 ppm P without disturbing the potato yield biomass indicating influence of a specific stress. This assumption is supported by a down-regulation of StCytc1 but an up-regulation of StAOX2 (comparing M/NM plants in Experiment 3). Then, ABA data suggest that a higher mycorrhizal colonization is the consequence of an initial physiological stress state implemented in plant prior to root contact by AMF, in which AOX and fermentation seems to take place. This connection between AOX-metabolism and fermentation leads to the hypothesis that AMF might partly stimulate plants to provide – or generate a metabolic environment that favors the fungal synthesis of – fermentation products as possible carbon sources for its storage needs. This assumption seems, to our knowledge, underestimated since no publication discusses directly these aspects on AMF, although it is known that presence of fermentative organisms/fermented products in soil can stimulate mycorrhizal development ( Medina et al., 2007 , 2010 ; Azcón et al., 2013 ), providing a clue. In particular, our data suggest that lactate, as one energy source for fungal needs, seems to be a good candidate, involved perhaps in sporulation potentials and arbuscule formation. Attempting to give a little more insight, first tests using exogenous lactate on potato plants under greenhouse conditions showed that it was possible to increase significantly the mycorrhizal colonization and arbuscule intensity associated with a positive MGD (Mercy, personal observation). As a remark, attention should be paid in further works to the role of the alternative glycolytic pathway (NAD/NADP malic enzymes) in mycorrhizal behavior, as it involves malate as source for fermentation and AOX pathway, both engaged during P-deficiency, a stress condition usually favoring the mycorrhizal development. Open Questions and Perspectives This is the first work that extensively studies phenotypic patterns by using respiratory chain inhibitors in mycorrhizal symbiotic associations. Plant growth parameters suggest that “AOX plants” (plants that are able to engage AOX pathway to a higher extent) are more positively responsive to AMF and have higher potentials in carbon allocation to the potato tuber than “COX plants” (plants that show a higher engagement of COX pathway), and plants treated with ABA or with KCN indicated that AMF inoculation is efficient to counteract extreme stresses in terms of growth performance. Under stress-related physiology, AOX might represent the main (or a more engaged) respiratory chain pathway in plant roots, concomitant with higher fermentation that might favor arbuscule development. In analogy to previous observations ( Liu et al., 2015 ), we observed that the presence of AMF reduces plant stress and seems to favor the COX pathway, which is consistent with higher energetic metabolism that associates to positive MGD. Therefore, artificial soft and transitory induction techniques of AOX metabolism in plants, without disturbing COX potential, could represent a physiological target for producers for improving mycorrhizal plant susceptibility and mycorrhizal dependency. It is very likely that the modulation of respiration partitioning occurs at different stages of AMF life cycle, as shown during presymbiotic phase by the induction of mitochondrial respiration by several plant root exudates ( Tamasloukht et al., 2003 ; Besserer et al., 2008 ) and AOX expression along the symbiotic establishment ( Campos et al., 2015 ). Optimal mycorrhizal development needs obviously oxygen (KCN data) but seems also related to fermentation (ABA data). This opens the possibility that AMF, at a whole, might belong to obligate aerobic fermentative organisms. Therefore, the role of fermentation in mycorrhizal behavior deserves further investigations providing potentially exciting new avenues for next studies. While the application of respiratory inhibitors induced significant changes from phenotypical parameters and the expression of some genes involved in transport and fermentation, interpretation of transcriptomic data related to AOX and COX pathways was challenging. However, respiration genes are hierarchically superimposed to specific metabolic pathway genes. Increasing data supports the hypothesis that the most crucial responses of AOX genes to changing environments are induced early during cell reprogramming before specific pathways are initiated and interfere with growth performance ( Arnholdt-Schmitt et al., 2006 ). Recently, this was confirmed for the carrot anti-freezing protein gene under cold treatment ( Campos et al., 2016 ). Furthermore, in A. thaliana , it was shown that limitation of the capacity of the alternative-, cytochrome-, or both termini-oxidases results in unique and overlapping transcriptional responses depending on growth conditions. Nevertheless, this research presented the first clues for the implication of respiratory metabolism in a mycorrhizal symbiosis seen as a holobiont level system. Thus, our research supports further deeper molecular-physiological studies on respiration traits and exploiting genetic approaches through overexpressing and knock-out or respiratory gene editing techniques. However, final progress in order to associate complex respiration metabolism to robust growth performance will absolutely depend also on the development of adequate predictive and phenotyping-screening technologies ( Fiorani and Schurr, 2013 ; Arnholdt-Schmitt et al., 2014 , 2016 ; Nogales et al., 2016 )."
} | 11,765 |
36590862 | null | s2 | 6,114 | {
"abstract": "All systems for processing signals, both artificial and within animals, must obey fundamental statistical laws for how information can be processed. We discuss here recent results using information theory that provide a blueprint for building circuits where signals can be read-out without information loss. Many properties that are necessary to build information-preserving circuits are actually observed in real neurons, at least approximately. One such property is the use of logistic nonlinearity for relating inputs to neural response probability. Such nonlinearities are common in neural and intracellular networks. With this nonlinearity type, there is a linear combination of neural responses that is guaranteed to preserve Shannon information contained in the response of a neural population, no matter how many neurons it contains. This read-out measure is related to a classic quantity known as the population vector that has been quite successful in relating neural responses to animal behavior in a wide variety of cases. Nevertheless, the population vector did not withstand the scrutiny of detailed information-theoretical analyses that showed that it discards substantial amounts of information contained in the responses of a neural population. We discuss recent theoretical results showing how to modify the population vector expression to make it 'information-preserving', and what is necessary in terms of neural circuit organization to allow for lossless information transfer. Implementing these strategies within artificial systems is likely to increase their efficiency, especially for brain-machine interfaces."
} | 408 |
37965057 | PMC10641440 | pmc | 6,115 | {
"abstract": "Polyhydroxyalkanoates (PHAs) have emerged as an environmentally friendly alternative to conventional polyesters. In this study, we present a comprehensive analysis of the genomic and phenotypic characteristics of three non-model thermophilic bacteria known for their ability to produce PHAs: Schlegelella aquatica LMG 23380 T , Caldimonas thermodepolymerans DSM 15264, and C. thermodepolymerans LMG 21645 and the results were compared with the type strain C. thermodepolymerans DSM 15344 T . We have assembled the first complete genomes of these three bacteria and performed the structural and functional annotation. This analysis has provided valuable insights into the biosynthesis of PHAs and has allowed us to propose a comprehensive scheme of carbohydrate metabolism in the studied bacteria. Through phylogenomic analysis, we have confirmed the synonymity between Caldimonas and Schlegelella genera, and further demonstrated that S. aquatica and S. koreensis , currently classified as orphan species, belong to the Caldimonas genus.",
"conclusion": "5 Conclusions In conclusion, the genomic and phenotypic characterization of the non-model bacteria Schlegelella aquatica LMG 23380 T and Caldimonas thermodepolymerans DSM 15264 and LMG 21645 provides valuable insights into microbial production of polyhydroxyalkanoates, sustainable and environmentally friendly polyesters. The genome assembly and functional annotation of these bacteria confirm their potential with regards to PHA production and reveal their individual characteristics. Particularly, the unique xyl operon present only in C. thermodepolymerans suggests their strong potential for biotechnological PHA production from xylose-rich resources. Furthermore, the fact that the strains of C. thermodepolymerans are also capable of utilizing cellobiose is of biotechnological interest since cellobiose is commonly present in lignocellulose-based media when enzymatic hydrolysis of cellulose is employed. Further, the fact that the studied strains of C. thermodepolymerans are capable of cellobiose utilization also indicates that the low efficiency of glucose metabolism might be linked to difficulties with the transport of glucose into the cells rather than a deficiency in its metabolization in the cells. In this case, glucose uptake and conversion into PHA might be potentially improved by introducing heterologous powerful glucose transporter using approaches of synthetic biology. Therefore, further investigation is required to fully understand the organism's properties and improve its potential for PHA production in agreement with the concept of NGIB.",
"introduction": "1 Introduction Polyhydroxyalkanoates (PHAs) are polyesters accumulated by numerous prokaryotes in the form of intracellular granules to serve as carbon and energy storage materials and to enhance stress robustness of bacterial cells [40] . Moreover, PHAs have emerged as environmentally friendly substitutes for petroleum-based polymers, offering a sustainable, renewable, biodegradable, compostable and also biocompatible alternative [23] , [48] . Although the production of bioplastics is seen as the way of the future and an integral part of the circular economy, less than 1% of total plastics production comes from the bioplastics industry [50] . Extremophiles are organisms able to survive and even prosper in extreme conditions such as acidic or basic pH levels, the presence of toxic elements, and immoderate temperatures [47] , [34] . Since the cultivation of extremophiles can be processed in a semi-sterile or even unsterile mode, which significantly lowers the cost of biotechnological procedures, these organisms have gained a key role in the so-called “Next-generation Industrial Biotechnology” (NGIB) concept [9] in recent years [11] . Further benefits come from the employment of thermophiles, bacteria that thrive at temperatures above 45 °C. Although biotechnological processes operate at high temperatures, they can be energetically feasible because the metabolic heat and energy released during mixing can be used to heat the bioreactor. In addition, cooling costs are modest because ambient air can be used to cool the process [20] , [42] . Several potent thermophilic PHAs producers can be found in the recently proposed family Sphaerotilaceae, which, after the taxonomic revisions based on phylogenomic comparisons, contains several genera, including the genus Caldimonas \n [31] . The most promising group of organisms within this genus is formed by species belonging originally to the genus Schlegelella \n [39] with the type species Schlegelella thermodepolymerans , which was initially studied for its ability to degrade PHAs such as 3-hydroxybutyrate and 3-mercaptopropionate copolymers [14] . Moreover, an extraordinary ability of the type strain S. thermodepolymerans DSM 15344 T to utilize xylose and synthesize 3-hydroxybutyrate and 3-hydroxyvalerate copolymers was recently reported by our group [26] and the bacterium was identified as a promising candidate for industrial production of PHAs from various xylose rich lignocellulose-based resources [25] . Subsequently, we provided the first complete genome assembly and the functional annotation of S. thermodepolymerans DSM 15344 T \n [37] . With the availability of complete genome sequences, Schlegelella thermodepolymerans was found to be a homotypic synonym of Caldimonas thermodepolymerans \n [31] . Since the genomes of the two other Schlegelella species were not available and 16S rRNA gene phylogeny was found to be insufficient to infer evolutionary relationships in the family Sphaerotilaceae , the two orphaned species, Schlegelella aquatica and Schlegelella koreensis , remained in the genus Schlegelella. However, additional research is needed to gain a comprehensive understanding of the evolutionary relationships within the genus, as well as to uncover the full genomic, metabolic, and biotechnological potential of these non-model bacteria. Here, we compared the newly assembled genome of the type strain of S. aquatica LMG 23380 T with the recently published genome of the type strain of S. koreensis ID0723 T and other genomes from the genus Caldimonas including two newly assembled genomes of non-type Caldimonas thermodepolymerans , formerly Schlegelella thermodepolymerans , species. Besides further revisiting their taxonomy, we aimed at their genotypic and phenotypic comparison because all Caldimonas / Schlegelella species remain underexplored, which prevents their possible use in industrial biotechnology. These comparisons included examining the substrate range and antibiotic susceptibility, or identifying genes for the key enzymes in core carbohydrate and PHA metabolism.",
"discussion": "4 Discussion Uncovering genomic and phenotypic traits with a focus on PHAs production by strains Schlegelella aquatica LMG 23380 T and Caldimonas thermodepolymerans DSM 15264 and LMG 21645 may provide important insights into microbial production of sustainable environmentally friendly polyesters. Based on our previous studies, C. thermodepolymerans can be considered a promising candidate for PHA production, especially from xylose-rich lignocellulose-based resources [25] , [26] . Since the genomic information was not previously available, genome assembly was a necessary first step for further studies. De novo genome assembly using ONT reads identified one circular contig in each bacterium, indicating the absence of plasmids and thus the presence of chromosomal DNA only. Furthermore, high-quality short Illumina reads were used to polish the assemblies. The mapping of nearly all of the short reads to the final genome, as well as the prediction of the replication origin oriC, were unambiguous, which confirms the genome had been assembled correctly. Although genome length and GC content are higher than the average for Gram-negative bacteria [30] , the values are consistent with the assumption that GC content is positively correlated with genome length [60] and with growth temperature [19] as well. Functional annotation, including the study of bacterial characteristics such as the function of individual genes, the ability to defend against foreign DNA, antibiotic resistance, and the requirements for genome editing, is the key prerequisite for understanding and manipulating the studied bacteria. Genes classified according to clusters of orthologous groups (COGs) revealed around 85% of gene functions in all of the studied Caldimonas / Schlegelella . However, the remaining 15% remains unknown, highlighting the uniqueness of these bacteria. Furthermore, the classification confirmed the genomic similarity among the C. thermodepolymerans strains and indicated a partial divergence between S. aquatica and C. thermodepolymerans based on different distributions across specific COGs within a narrow range. The prediction of Restriction-Modification (R-M) systems revealed the diversity of this cellular defense mechanism in the studied bacteria. The lack of active R-M systems was registered in S. aquatica LMG 23380 T genome, with only a partial type I system being identified, suggesting its inactivity. In contrast, all of the studied C. thermodepolymerans possess at least one complete R-M system, indicating a strong system protecting C. thermodepolymerans strains from foreign DNA, which may pose challenges in genome editing [45] . Regarding further potential opportunities for future genome engineering, all of the studied bacteria were found to have the ability to receive foreign DNA as they contain CRISPR arrays. These arrays can be also adopted as a reservoir of components for genome editing of Caldimonas strains and potentially other thermophilic bacteria [17] . Native CRISPR-Cas systems in strains S. aquatica LMG 23880 T and C. thermodepolymerans DSM 15264 and LGM 21645 will be further studied and can be repurposed for genome editing of these bacteria [59] . The observed susceptibility of all Caldimonas strains to four out of the five selected antibiotics will facilitate their future genetic engineering as various selection markers can be used. Identified antibiotic-resistance genes can be subsequently removed to prevent a potential outbreak of antibiotic resistance. In the genomic era, a polyphasic approach combining examinations of genotypic and phenotypic traits is still necessary and has been practiced for more than a decade in prokaryotic taxonomy [55] . Here, the classification of the studied bacteria using a combination of digital DNA-DNA hybridization (dDDH) and phylogenomic analysis confirmed that the newly analyzed non-type strains C. thermodepolymerans DSM 15264 and LMG 21645 belong to C. thermodepolymerans species and are very similar to the type strain C. thermodepolymerans DSM 15344 T . Additional cultivation experiments also showed that these strains share unique phenotypic traits, particularly a preference for xylose over glucose, which seems to be a unique feature of C. thermodepolymerans that cannot be found in other species of the genus Caldimonas . This suggests, along with the fact that C. thermodepolymerans , formerly Schlegellela thermodepolymerans , was the type species of the genus Schlegelella covering four species, that the genus Caldimonas / Schlegelella is much more diverse than previously assumed. The previous absence of representative genomes of S. aquatica and S. koreensis , coupled with the limitations of the 16S rRNA gene in inferring the phylogeny within the family Sphaerotilaceae \n [31] prevented their accurate taxonomic placement. An analysis of the newly assembled S. aquatica and publicly available S. koreensis genomes revealed that Schlegelella aquatica and Schlegelella koreensis are homotypic synonyms for Caldimonas aquatica and Caldimonas koreensis , respectively. At the same time, genotypic analysis proved that both organisms represent separate species, as also shown by their different phenotypes. S. aquatica , in comparison to C. thermodepolymerans , exhibits slower growth and lower production of PHAs when utilizing various carbon sources and prefers glucose over xylose. Although we did not analyze the phenotype of S. koreensis , this species had been already shown to possess unique traits such as different temperature ranges for growth and the inability to utilize various saccharides while still producing PHA granules [8] . PHAs can be synthesized through four different pathways [52] . In the tested strains the most common metabolic a three-step pathway, encoded by the genes forming the phaCAB operon [26] , [56] was identified, this synthetic route utilizes predominantly acetyl-CoA as a primary substrate. This key metabolic intermediate is converted into acetoacetyl-CoA, followed by the conversion into 3-hydroxybutyryl-CoA [38] , [44] . Given that the phaCAB operon has been identified in each of the studied Caldimonas strains, exploitation of this pathway during the growth on the assessed lignocellulosic sugars (xylose, glucose, and cellobiose) can be assumed ( Fig. 4 ). Additionally, PHA metabolism is closely linked with carbohydrate catabolism and fatty acids β-oxidation which provide precursors (especially acetyl-CoA) for PHA synthesis and fatty acid β-oxidation which can also supply suitable 3-hydroxyacids in various bacteria. Fig. 4 Schematic illustration of proposed carbohydrate metabolism in studied Caldimonas / Schlegelella strains. Xylose isomerase pathway (formed by XylA xylose isomerase and XylB xylulokinase) and the pentose phosphate pathway are shown using brown arrows, the Embden-Meyerhof-Parnas pathway is shown with orange arrows, and the Entner-Doudoroff pathway with magenta arrows. Abbreviations (enzymes): Eda, 2-keto-3-deoxy-6-phosphogluconate aldolase; Edd, 6-phosphogluconate dehydratase; Eno, phosphopyruvate hydratase; Fba, fructose-1,6-biphosphate aldolase; Fbp, fructose-1,6-biphosphatase; Gap, glyceraldehyde-3-phosphate dehydrogenase; Gcd, glucose dehydrogenase; Gnd, 6-phosphogluconate dehydrogenase; Pdh, pyruvate dehydrogenase; Pgi, glucose-6-phosphate isomerase; Pgk, phosphoglycerate kinase; Pgl, 6-phosphogluconolactonase; Pgm, phosphoglycerate mutase; PhaA, acetyl-CoA acetyltransferase (3-ketothiolase); PhaB, acetoacetyl-CoA reductase; PhaC, poly(3-hydroxyalkanoate) polymerase; Pyk, pyruvate kinase; Rpe, ribulose-5-phosphate 3-epimerase; RpiA, ribose-5-phosphate isomerase; Tal, transaldolase; Tkt, transketolase; Tpi, triosephosphate isomerase; Zwf glucose-6-phosphate dehydrogenase. Abbreviations (metabolites): AA-CoA, acetoacetyl coenzyme A; Acetyl-CoA, acetyl coenzyme A; DHPA, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; FBP, fructose 1,6-biphosphate; F6P, fructose 6-phosphate; GLL, glucono-δ-lactone; GLN, gluconate; G3P glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; 3HB-CoA, 3-hydroxybutyryl coenzyme A; KDPG, 2-keto-3-deoxy-6-phosphogluconate; 2KG, 2-ketogluconate; 2KG-6 P, 2-ketogluconate 6-phosphate; NADH, reduced nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; 6PG, 6-phosphogluconate; PHB, poly-3-hydroxybutyrate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, seduheptulose 7-phosphate; XLN, xylonate; X5P, xylulose 5-phosphate. GtsABCD and XylFGH stand for mannose/glucose and xylose ABC transporter, respectively. Note that xylose isomerase pathway and b-glucosidase are not present in S. aquatica . Fig. 4 Genes involved in these three pathways have been identified in all the bacteria studied, which likely implies the ability to synthesize PHAs by all the pathways mentioned; however, further research is needed to confirm this hypothesis. In addition, several differences between S. aquatica ( Sa ) and C. thermodepolymerans ( Ct ) have been identified. Firstly, S. aquatica most likely does not possess the enzymatic apparatus for extracellular degradation of PHA materials present in the environment. On the other hand, C. thermodepolymerans was found to possess highly effective extracellular PHA depolymerase. In fact, the great PHA degradation efficiency is one of the most notable features of C. thermodepolymerans , which is also reflected in its taxonomic name: thermodepolymerans – i.e. capable of degradation of PHA polymers under thermophilic conditions [14] . Further, a gene belonging to the polyhydroxyalkanoic acid group family with unknown function is exclusively present in the genome of Sa . Genomic differences were also confirmed by the cultivation experiments. The ability to produce PHA was demonstrated in all of the investigated strains. However, a significantly lower ability to utilize carbohydrate substrates and produce PHA was observed in S. aquatica LMG 23380 T . C . thermodepolymerans DSM 15264 showed the greatest similarity to the type strain C. thermodepolymerans DSM 15344 T . A high increase in biomass was observed for both cellobiose and xylose substrates. The contrasting lack of growth and PHA formation in S. aquatica on xylose and cellobiose can be explained by the absence of the xyl operon and the β-glucosidase gene in its genome. Unlike S. aquatica , all Ct strains possess these genes, as reflected in their ability to utilize xylose and cellobiose. It is not clear now why all Ct strains grow much worse on glucose than on cellobiose. Since all the bacteria investigated in this study possess genes assumed to encode metabolic traits that ensure glucose utilization ( Fig. 4 ), we hypothesize that the bottleneck limiting the utilization of this sugar may lie in its transport across the cytoplasmic membrane. The identified GtsABCD glucose/mannose transporter might have a higher affinity for cellobiose than for monomeric glucose, whose transport would be either slow ( Sa LMG 23380 T , Ct DSM 15344 T ) or negligible ( Ct DSM 15264 and Ct LMG 21645). Uptake of cellobiose and other cellooligosaccharides through the ABC-type glucose transporters is common in bacteria [13] , [43] . The transport of oligosaccharides is more economical with regards to ATP than glucose transport. Our hypothesis is further supported by the presence of the β-glucosidase gene in the gts operon of Ct strains and by the fact that residual glucose is often detected in culture supernatants of the strains grown on cellobiose (data not shown). The latter observation also implies that at least a part of the cellobiose substrate is cleaved to glucose monomers outside the cell. A signal sequence specific for the twin-arginine translocation (Tat) pathway was identified at the 5 ´ end of the β-glucosidase genes in the Ct strains. Since the Tat pathway is known to translocate already folded (and active) proteins across the cytoplasmic membrane [4] , we argue that cellobiose hydrolysis by β-glucosidase in the Ct strains studied takes place both in the cytoplasm and outside the cell ( Fig. 4 ). The proposed scheme of the upper carbohydrate metabolism in the studied thermophilic PHA producers ( Fig. 4 ) suggests that all three tested lignocellulosic sugars can be utilized, despite the lack of pgl and gnd homologs in the Caldimonas genomes as hydrolysis of 6-phosphogluconolactone can occur spontaneously [16] . A possible compensatory mechanism for the lack of Gnd is gluconate 6-phosphate undergoing the ED shunt to glyceraldehyde 3-phosphate and pyruvate. This way, 6-phosphogluconate would not become a dead-end product and at least 1 mol of NADPH per 1 mol of glucose would be produced in the glucose-6-phosphate dehydrogenase (Zwf) reaction in the oxidative branch of the PPP. During the growth on glucose or cellobiose, at least a small reverse flux from glyceraldehyde 3-phosphate and fructose 6-phosphate to the non-oxidative PPP is required to ensure the formation of nucleotide precursors. Hence, the preference of xylose over glucose in Caldimonas strains might be explained by the fact that the pathway for securing important metabolic precursors in PPP such as ribulose 5-phosphate is shorter with pentose. However, more experimental data is necessary to investigate this phenomenon."
} | 5,073 |
21966901 | null | s2 | 6,117 | {
"abstract": "Bacteria employ a variety of mechanisms to promote and control colonization of their respective hosts, including restricting the expression of genes necessary for colonization to distinct situations (i.e. encounter with a prospective host). In the symbiosis between the marine bacterium Vibrio fischeri and its host squid, Euprymna scolopes, colonization proceeds via a transient biofilm formed by the bacterium. The production of this bacterial biofilm depends on a complex regulatory network that controls transcription of the symbiosis polysaccharide (syp) gene locus. In addition to this transcriptional control, biofilm formation is regulated by two proteins, SypA and SypE, which may function in an unusual regulatory mechanism known as partner switching. Best characterized in Bacillus subtilis and other Gram-positive bacteria, partner switching is a signalling mechanism that provides dynamic regulatory control over bacterial gene expression. The involvement of putative partner-switching components within V. fischeri suggests that tight regulatory control over biofilm formation may be important for the lifestyle of this organism."
} | 285 |
39530358 | PMC11697164 | pmc | 6,122 | {
"abstract": "Abstract Urea is hypothesized to be an important source of nitrogen and chemical energy to microorganisms in the deep sea; however, direct evidence for urea use below the epipelagic ocean is lacking. Here, we explore urea utilization from 50 to 4000 meters depth in the northeastern Pacific Ocean using metagenomics, nitrification rates, and single-cell stable-isotope-uptake measurements with nanoscale secondary ion mass spectrometry. We find that on average 25% of deep-sea cells assimilated urea-derived N (60% of detectably active cells), and that cell-specific nitrogen-incorporation rates from urea were higher than that from ammonium. Both urea concentrations and assimilation rates relative to ammonium generally increased below the euphotic zone. We detected ammonia- and urea-based nitrification at all depths at one of two sites analyzed, demonstrating their potential to support chemoautotrophy in the mesopelagic and bathypelagic regions. Using newly generated metagenomes we find that the ureC gene, encoding the catalytic subunit of urease, is found within 39% of deep-sea cells in this region, including the Nitrososphaeria (syn., Thaumarchaeota ; likely for nitrification) as well as members of thirteen other phyla such as Proteobacteria , Verrucomicrobia , Plantomycetota , Nitrospinota , and Chloroflexota (likely for assimilation). Analysis of public metagenomes estimated ureC within 10–46% of deep-sea cells around the world, with higher prevalence below the photic zone, suggesting urea is widely available to the deep-sea microbiome globally. Our results demonstrate that urea is a nitrogen source to abundant and diverse microorganisms in the dark ocean, as well as a significant contributor to deep-sea nitrification and therefore fuel for chemoautotrophy.",
"introduction": "Introduction Nitrogen (N) is an essential nutrient for all living organisms [ 1 ]; however, bioaccessible N can be a scarce and therefore limiting element in marine environments [ 2 ]. Ammonium and nitrate are among the most important forms of nitrogen in the oceans. While ammonium is assimilable by most microorganisms, nitrate must be enzymatically reduced to ammonium before assimilation, incurring an energetic cost and excluding organisms without this enzymatic machinery [ 3 , 4 ]. Ammonium is therefore typically preferred, and is generally scarce below the euphotic zone (low nM range) [ 5 ] while nitrate concentrations can be orders of magnitude higher, especially at depth [ 6 , 7 ]. Some microorganisms also use inorganic nitrogen as electron acceptors or donors in respiratory processes, increasing the demand for nitrogen in the environment. For example, ammonia can be oxidized to nitrite by chemoautotrophic ammonia-oxidizing archaea (AOA; i.e. Nitrososphaeria , syn., Thaumarchaeota ) and ammonia-oxidizing bacteria (AOB) [ 8 , 9 ]. N use in general, and ammonium use in particular, connects closely with carbon cycling, as its availability can influence rates of both heterotrophic [ 10 ] and photo/chemo-autotrophic activity [ 11–13 ]. N dynamics have been studied extensively in the euphotic zone (e.g. [ 5 , 14 ]), but less is known about nitrogen cycling in the deep sea, a region increasingly recognized as hosting a diverse, active, and influential microbiome [ 7 , 15 , 16 ]. Urea, a form of organic nitrogen which can be cleaved enzymatically to create two molecules of ammonia, has been proposed as a key substrate for both anabolism and nitrification in the deep sea [ 17 , 18 ]. As a source of energy for chemoautotrophy, urea-based nitrification could support organic matter production at depth, thereby ameliorating current discrepancies in the oceanic carbon cycle [ 19 ]. However, experimental evidence regarding the abundance [ 20 , 21 ] and use of urea in the meso- and bathypelagic is still rare or lacking, respectively. Nitrososphaeria -affiliated ureC genes and transcripts (encoding the catalytic subunit of urease) have been detected in the epipelagic [ 17 ], mesopelagic [ 19 , 22–24 ] and bathypelagic [ 15 , 25 ], suggesting the ability of nitrifying archaea to utilize this substrate through the entire water column. Supporting this, urea-based nitrification has been measured at the ocean surface [ 17 ], at the base of the epipelagic (at 150 m [ 26 , 27 ]), and within the mesopelagic (to 300 m [ 24 ] and to 1000 m [ 28 ])—at rates comparable to those for ammonia. Similarly, urea assimilation is extensive in the surface ocean [ 29 ], and has been implicated in the mesopelagic based on the observation of urea degradation at rates exceeding calculated N demand for nitrification [ 30 ]. However, direct measurements of urea assimilation have not been made in the mesopelagic or bathypelagic, measurements of urea oxidation are missing in the bathypelagic, and the prevalence and phylogenetic diversity of organisms containing ureC in the aphotic ocean have not been determined. Therefore, whether the ability to cleave urea is common or rare in the deep sea, taxonomically or numerically, is still unknown, and leaves the accessibility of this potentially large source of nitrogen and energy unconstrained. In this work, we assessed the role of urea in sustaining microbial biomass production and nitrification from 50 to 4000 m water depth in the northeast Pacific Ocean. We start with an investigation of urea concentrations with depth at six sites across a 300 km transect. At two of these sites, one at the base of the continental slope (“Slope site”) and one at the far end of the transect (“Open Ocean site”), we use incubation experiments with 13 C 15 N-urea and single-cell analysis by nanoscale secondary ion mass spectrometry (nanoSIMS) to determine the proportion of cells assimilating urea-derived nitrogen, and at what rates. We use these same incubations to determine urea- and ammonia- based nitrification rates to assess their role in microbial catabolism throughout the water column. Indeed, although genomic evidence for ammonia-based nitrification at depth is convincing [ 16 , 31 ], even ammonia-based nitrification has not been experimentally confirmed below the mesopelagic. We generated thirteen deeply sequenced metagenomes throughout the Slope and Open Ocean sites, and together with public metagenomes from around the world assess the distribution of the ureC gene and the potential role of specific taxa in the utilization and recirculation of urea. Finally, we used the combined ammonia- and urea-based nitrification rates to estimate deep-sea carbon fixation rates, and compare these to estimated rates of sinking particulate organic carbon (POC) to estimate the significance of nitrification-based chemoautotrophy at these sites. Together, our lines of inquiry demonstrate the use of urea-derived nitrogen in both microbial anabolism and catabolism in the deep northeastern Pacific Ocean, with implications for nitrogen and carbon cycling globally.",
"discussion": "Discussion Urea is increasingly recognized as a source of nitrogen for cell growth [ 29 , 52 , 53 ] and nitrification [ 17 , 18 ] in the sunlit ocean. In the euphotic zone, nitrogen from urea is assimilated by phylogenetically diverse taxa, including Cyanobacteria , Proteobacteria , and Nitrososphaerota (e.g. Nitrososphaeria ) [ 14 , 18 , 19 , 26 , 53 , 54 ], at rates exceeding those for nitrate, leucine, glutamate [ 14 ], and even ammonium [ 55 ]; it is also oxidized by nitrifying Nitrososphaeria to support chemoautotrophy [ 56 , 57 ]. Our observations in the northwest Pacific Ocean indicate that the significance of this molecule extends to the aphotic zone—where an equally broad yet predominantly different set of organisms cleave it—and indicate that its role may be even more central to ecosystem functioning there than in surface waters. Our data show that both the concentration of urea and the relative abundance of ureC genes increase with depth, as does the microbial preference for it as a nitrogen source over ammonia. Additionally, while rates of urea-based nitrification are lower at depth than at the surface, they are comparable to that of ammonia in both realms, and likely play an outsized role in microbial community dynamics at depth by supporting the production of organic matter in a more energy- and carbon-limited system than at the surface. We found that urea-derived nitrogen is widely and extensively assimilated by microorganisms in the aphotic zone. We detect assimilation of urea-derived nitrogen in 25% of cells on average in the meso- and bathypelagic regions. However, since many cells in these regions are below our detection limit of anabolic activity [ 7 ], it is possible that we are underestimating the proportion of cells assimilating urea. Using ammonium uptake as a proxy for detectable anabolic activity [ 7 , 58 ] we estimate that 60% of detectably active cells in the meso- and bathypelagic assimilate urea-derived nitrogen. Cross-feeding of 15 N-labelled substrates can cause these proportions to be greater than the number of cells directly consuming urea, and for this reason, we refer to the assimilation of 15 N in the 15 N-urea incubations as assimilation of “urea-derived” nitrogen. However, the distinct trends between urea and ammonium ( Fig. 2 ), suggest that cross-feeding was minimal and direct use of urea is the dominant process underlying our observations. Additionally, the paired metagenomic data is roughly consistent with the uptake data; we estimate that an average of 39% of cells in the meso- and bathypelagic at these sites contain a ureC gene (as described in more detail below). Regardless of what proportion of the assimilation was directly from urea versus recycled substrates, the widespread and high rates of consumption of urea-derived N indicates that the large reservoir of urea-nitrogen in the deep sea—on average an order of magnitude more abundant than ammonium—is available to most cells. Nitrate remains the largest pool of fixed nitrogen in the deep sea, averaging over two orders of magnitude more abundant than urea at our study sites. Our observations of urea assimilation occurred in the presence of these high concentrations of nitrate, suggesting a preference for urea over nitrate. Indeed, we found that ureC genes were more abundant than those related to assimilatory nitrate reduction (such as nasA ) within our study sites, as well as a broad distribution of publicly available deep sea datasets ( Fig. S3 ). Previous work has also reported relatively low detection of nasA in the Malaspina global deep-sea metagenomic dataset [ 15 ]. Preference for urea is likely related to the higher energy requirements of the assimilatory reduction of nitrate [ 59 , 60 ], a difference that might be particularly relevant in the energy-poor aphotic zone. While gene abundances are useful indicators of potential activity, and how well a given ability is distributed across a community, direct comparisons of the uptake of nitrate and urea in the deepest regions of the oceans would be beneficial to directly compare the proportions of cells capable of assimilating each, and with what preference. The meso- and bathypelagic regions accounted for nearly half of the total pelagic urea assimilation in the Slope site—more than it contributed to either ammonium or amino acid assimilation [ 7 ]—indicating that urea is a more important nitrogen source in the deep sea relative to the surface than for either ammonium or amino acids. Urea also represents a major potential substrate for nitrification by members of the Nitrososphaeria [ 17–19 , 24 ]. Remarkably, urea-based nitrification can also happen in the presence of substantial ammonia [ 17 ], suggesting that urea is not only an alternative when ammonia is scarce. Furthermore, a recent study shows that some AOB repress the use of extracellular ammonia in the presence of ammonia derived from urea hydrolysis in the cytoplasm [ 55 ]. While previous studies have highlighted the significance of urea-driven nitrification in the epipelagic [ 17 , 18 , 61 ] and mesopelagic regions [ 24 , 28 ], urea-driven nitrification has not been directly measured in the bathypelagic region. The detection of urea-based nitrification at all depths of our Slope site suggests that bathypelagic nitrifiers can indeed use urea as a substrate ( Fig. 3 ). Oxidation of ammonia after urea hydrolysis by other community members is also possible, but even in this case, this confirms that urea-derived nitrogen is readily available to microbes for nitrification. Direct uptake and hydrolysis of urea by deep-sea nitrifiers is supported by the metagenomic analysis, which showed extensive genetic potential for urea use by nitrifiers: ureC genes were found within Nitrososphaeria MAGs and over half of sequencing coverage of ureC -containing contigs in the meso- and bathypelagic was affiliated with Nitrososphaeria ( Fig. 5A ). The rates of urea-based nitrification were statistically indistinguishable from those for ammonium at all depths, consistent with the previous work in the mesopelagic [ 24 , 28 ], indicating a potentially significant role for urea in deep-sea nitrification. Rates of both ammonia- and urea-based nitrification decreased with depth and distance from shore ( Fig. 3 ), consistent with the trends we observed in overall anabolic activity previously at this site [ 7 ]. Using metagenomics, we determined both the distribution of ureC in microbial communities at our study site and in globally sourced datasets, and also classified the taxa containing ureC . We detected ureC genes throughout the water column and found that their prevalence within the community—the proportion of microbial cells possessing it in a given sample—reached a maximum in the aphotic zone in both our study site and the other global datasets we analyzed. This is consistent with a previous analysis that found that the prevalence of ureC within Thaumarchaeota increased with depth in both Artic and Antarctic regions [ 19 ], as well as a recent proteomics study which observed peak relative abundance of urease in the bathypelagic region of the global ocean [ 62 ]. Overall, we see that about a third of the cells in the dark ocean (average 39% in our dataset, and average of 30% in the global datasets) contain ureC . Both the contig- and MAG-based analyses identified diverse taxa containing ureC genes at our site, with fourteen distinct phyla identified by the former and 11 by the latter. The MAG-based taxonomic identification of ureC -containing genomes is more robust than the contig-based identifications due to the greater sequence length available for consideration and less vulnerability to misclassifications due to horizontal gene transfer. However, the contig-based approach provides a more comprehensive overview of the community (accounting for, on average, 32% more of the total metagenomic reads than the MAG set; Fig. S4 ), and includes taxa that systematically evade genomic binning. Notably, 11 of the fourteen phyla identified in the contig-based analysis were also identified with the MAG-based analysis. The groups identified as containing ureC in the 50 m samples are generally consistent with previous work in the euphotic zone, especially in the identification of Gammaproteobacteria [ 22 ] and Prochlorococcus [ 14 ]. The deep-sea analysis revealed that some taxonomic groups with members known to use urea at the surface also have members with the genetic capacity to do so at depth, including Nitrososphaeria ( Nitrososphaerales ), Verrucomicrobiota , and Myxococcota , as well as members of several groups not before reported to utilize urea, including SAR202 and alphaproteobacterial TMED109. We interpret the presence of ureC genes in taxa not known to oxidize ammonia, and in MAGs without an amoA gene, as evidence of potential urea use for nitrogen acquisition. Conversely, when found together with amoA (i.e. within the Nitrososphaeria ), it may be used for both nitrogen acquisition for biomass and for nitrification. While our metagenomic analysis is consistent with a large role for urea in nitrifying organisms in the deep sea—evidenced by the large fraction of ureC genes within the Nitrososphaeria —our work also highlights the wide diversity of organisms capable of cleaving it. As not all nitrifiers contain urease (e.g. Nitrosopelagicus brevis CN25 [ 63 ], Nitrosopumilus maritimus [ 23 ]), there may be an important relationship between heterotrophic urea degraders and chemoautotrophic nitrifiers, with ammonia shared in one direction and organic carbon (and/or other metabolites [ 64 ]) in the other. This is similar to the exchange of ammonia for nitrite previously suggested between ureC -containing nitrite-oxidizing bacteria and archaeal nitrifiers [ 54 ]. The implications of deep-sea ammonia- and urea-based nitrification on the marine carbon cycle are considerable. It is often assumed that the main—and essentially only—source of organic carbon to the dark ocean is gravitational POC [ 65 ]. However, the persistent imbalance between known supply and demand of organic matter in the deep sea highlights the inadequacies of this perspective [ 66 ]. The potential for endogenous production of organic carbon (e.g. chemoautotrophy) to contribute significantly to the deep-sea carbon budget is increasingly recognized [ 67 , 68 ], with regional measurements of dark DIC fixation equaling an estimated 15–53% of gravitational POC, and 12–72% of organic carbon demand (North Atlantic and Arctic Oceans [ 69–71 ]). Nitrite-oxidizing bacteria have been reported to contribute 15–45% of total inorganic carbon fixation in the mesopelagic North Atlantic based on microaudioradiography [ 54 ], but how specific organisms/metabolisms contribute to total chemoautotrophy is generally not well constrained. Rates of maximum potential DIC fixation by ammonia-oxidizing archaea have been previously approximated to be over an order of magnitude lower than total dark DIC fixation in the North Atlantic, but were calculated based on availability of ammonia, not direct measurements [ 69 ]. Our observations in the Northwest Pacific Ocean indicate that urea- and ammonia-based nitrification could support DIC fixation of 5 and 7% of the estimated gravitational POC entering the top of the mesopelagic at the Open Ocean and Slope sites, respectively. As total DIC fixation was not measured here, we cannot determine the contribution of these processes to total chemoautotrophy at these sites. However, these values are significant in comparison to the POC flux; gravitational POC fluxes and quality (bioaccessibility) decrease significantly with depth in the water column [ 72 , 73 ]. In contrast, organic carbon generated at depth is generally labile, highlighting the potential importance of even small amounts of endogenously produced organic carbon [ 74 ]. To fully assess the role of nitrification and chemoautotrophy more generally in helping balancing the carbon budget, more studies are required, including direct measurements of carbon fixation ideally at in situ pressures and concurrent measurement of sinking POC flux. However, our estimates provide evidence that the combination of urea- and ammonia-based nitrification can serve as a substantial source of endogenous organic carbon in the aphotic northeastern Pacific Ocean, and supports the notion that deep-sea chemoautotrophy should not be overlooked in models of the biological carbon pump. In summary, our study reveals a large reservoir of urea-N in the deep sea ( Fig. 1 ), widespread genetic potential for urea utilization in the meso- and bathypelagic ( Figs. 4 and 6 ), and direct evidence for both extensive assimilation of urea-derived nitrogen ( Fig. 2 ) and the persistence of both urea- and ammonia-based nitrification throughout the epi-, meso-, and bathypelagic ( Fig. 3 ). While additional direct measurements are necessary to confirm our results globally, we contend that urea use is likely widespread throughout the global deep sea on the basis of the generally physicochemically representative nature of our study site and the high proportions of ureC -encoding microorganisms throughout the global metagenomic datasets analyzed here. These results address long-standing hypotheses about the potential for urea to fuel nitrification in deep waters, and indicate the potential for chemoauototrophy at depth to significantly impact the marine carbon budget."
} | 5,132 |
37233284 | PMC10219321 | pmc | 6,123 | {
"abstract": "3-Hydroxypropionic acid (3-HP) is an important intermediate compound in the chemical industry. Green and environmentally friendly microbial synthesis methods are becoming increasingly popular in a range of industries. Compared to other chassis cells, Yarrowia lipolytica possesses advantages, such as high tolerance to organic acid and a sufficient precursor required to synthesize 3-HP. In this study, gene manipulations, including the overexpression of genes MCR-N C a , MCR-C C a , GAPN S m , ACC1 and ACS S e L 641 P and knocking out bypass genes MLS1 and CIT2 , leading to the glyoxylate cycle, were performed to construct a recombinant strain. Based on this, the degradation pathway of 3-HP in Y. lipolytica was discovered, and relevant genes MMSDH and HPDH were knocked out. To our knowledge, this study is the first to produce 3-HP in Y. lipolytica . The yield of 3-HP in recombinant strain Po1f-NC-14 in shake flask fermentation reached 1.128 g·L −1 , and the yield in fed-batch fermentation reached 16.23 g·L −1 . These results are highly competitive compared to other yeast chassis cells. This study creates the foundation for the production of 3-HP in Y. lipolytica and also provides a reference for further research in the future.",
"conclusion": "5. Conclusions In this paper, we obtained the recombinant Y. lipolytica Po1f-NC-14 by overexpressed genes MCR-N C a , MCR-C C a , GAPN S m , ACC1 and ACS S e L 641 P , knocking out genes MLS1 and CIT2 , leading to the glyoxylate cycle, and knocking out 3-HP degradation-related genes MMSDH and HPDH . The yield of 3-HP in shake flask fermentation reached 1.128 g·L −1 , and the yield in a fed-batch fermentation in a 1 L bioreactor reached 16.23 g·L −1 . We also analyzed the impact of changing fermentation conditions on 3-HP production and the tolerance of Y. lipolytica to the product. This study utilized Y. lipolytica to produce 3-HP, creating new directions of research and providing insights into the synthesis of organic acid in unconventional yeast, which will provide a reference for future studies.",
"introduction": "1. Introduction 3-Hydroxypropionic acid (3-HP, C 3 H 6 O 3 ) was listed as 1 of the 12 kinds of platform chemicals with the greatest potential by the US Department of Energy (DOE) in 2004. It has great application prospects and market value [ 1 ]. Many industry chemicals can be synthesized through diverse chemical reactions using 3-HP as material, such as malonic acid, 1,3-propanediol, acrylic acid, β -propiolactone, etc. [ 2 ]. Biodegradable plastics, including poly(3-hydroxypropionate) (P3HP) [ 3 ] and poly(3-hydroxybutyrate) (PHB) [ 4 ], can also be synthesized using 3-HP or its esterified derivatives. They have broad prospects in substituting for traditional petroleum-based plastics. Due to the high demand for 3-HP in the market, a high-yield 3-HP synthesis method is the current research focus. There are some general disadvantages in traditional chemical synthesis methods, such as the high cost of the starting materials and process, as well as the environmental incompatibility of the chemical approaches. Therefore, it is more suitable to obtain 3-HP using a biosynthesis method [ 5 ]. Researchers initially tried to synthesize 3-HP using prokaryote as a platform. Because of the clear genetic background and diversified gene manipulation tools, Escherichia coli became the first choice for the heterologous expression for production of 3-HP [ 6 ]. The Park research group expressed glycerol dehydratase (DHAB) from Klebsiella pneumonia and aldehyde dehydrogenase (ALDH) from Escherichia coli in E. coli BL21, and the initial yield of 3-HP was 0.58 g·L −1 by flask fermentation [ 7 ]. After optimizing the conditions in terms of pH, medium composition, dissolved oxygen, etc., the concentration of 3-HP reached 31 g·L −1 in a fed-batch culture using a 5 L bioreactor [ 8 ]. On this basis, the researchers switched the chassis strain to E. coli BL21(DE3), which had stronger tolerance to 3-HP and overexpressed glycerol dehydratase (DHAB) and its activators GdrA and GdrB from K. pneumoniae and α -ketoglutarate semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense . The yield of 3-HP was increased by 24.8% to 38.7 g·L −1 in a fed-batch culture [ 9 ]. With the premise of overexpressing the key rate-limiting enzyme, knocking out the bypass pathway genes can also increase the yield of target product. Another research work knocked out ACKA , PTA , YQHD , which are the key genes used to synthesize acetic acid and ethanol after expressing the mutated succinic semialdehyde dehydrogenase (GabD4 _E209Q/E269Q) from Cupriavidus necator and glycerol dehydratase (DHAB) from K. pneumoniae . The yield of 3-HP reached by 71.9 g·L −1 in a fed-batch culture [ 10 ], which was the highest yield of 3-HP synthesized in E. coli using glycerol as the substrate. Additionally, researchers tried to synthesize 3-HP through the malonyl-CoA pathway in E. coli . Malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus and acetyl-CoA carboxylase (ACC) from Corynebacterium glutamicum were expressed in E. coli , and the yield of 3-HP reached 10.08 g·L −1 after 36 h fermentation in a fed-batch culture [ 11 ]. In order to fundamentally improve the activity of the limiting enzyme, researchers found that the enzyme activity of malonyl-CoA reductase (MCR) from C. aurantiacus was severely inhibited under the condition of the bacterial culture temperature. The MCR was split into two different functional structures MCR-N and MCR-C to catalyze reactions individually. Finally, the yield of 3-HP reached 40.6 g·L −1 in a fed-batch culture [ 12 ]. Comparing to E. coli , K. pneumoniae has stable glycerol dehydratase (DHAB) and activators; it can also synthesize VB12, which is the key co-enzyme in the glycerol pathway. To explore gene modification from another research direction, researchers overexpressed aldehyde dehydrogenase (ALDH) from Pseudomonas sp. and used an appropriate promoter tac. The chassis cell was also knocked out the bypass gene LDH1 , LDH2 and PTA . The yield of 3-HP reached 83.8 g·L −1 3-HP [ 13 ]. They recruited three tandem repetitive tac promoters to overexpress an endogenous ALDH (PUUC) and obtained 102.6 g·L −1 3-HP, which was the highest yield reported so far [ 14 ]. The most significant problem with the prokaryotic production of 3-HP is that the prokaryotic chassis strain cannot tolerate low pH and product toxicity. Therefore, the researchers started performing the heterologous synthesis of 3-HP with yeast [ 15 ]. The researchers expressed the mutated acetyl coenzyme A (acetyl-CoA) carboxylase (ACC_S659A/S1157A) in Saccharomyces cerevisiae and preliminarily synthesized 3-HP; the yield was 0.28 g·L −1 [ 16 ]. On this basis, they expressed a series malonyl-CoA pathway key genes in S. cerevisiae , including MCR C a , ACC1 , ALD6 , ACE S E , ADH2 , and knocked out MLS1 which encodes cytosolic malate synthase; the yield of 3-HP reached 0.46 g·L −1 [ 17 ]. In another study, gene expression cassettes P T E F 1 BS123- MCR and P H X T 1 -FAS1 were constructed in S. cerevisiae . The malonyl-CoA sensor was developed based on the FapR transcription factor of Bacillus subtilis and applied in S. cerevisiae . The yield of 3-HP reached 0.8 g·L −1 [ 18 ]. In contrast with conventional metabolic regulation, for a different type of study, researchers designed a malonyl-CoA sensor in S. cerevisiae using an adapted bacterial transcription factor FapR and its corresponding operator fapO to gauge intracellular malonyl-CoA levels. Meanwhile, they co-overexpressed the two novel gene targets PMP1 and TPL1 , which were discovered by integrated sensor–genetic screening and achieved a concentration of 3-HP of 1.2 g·L −1 [ 19 ]. In addition, another research study expressed four heterologous genes in P. pastoris , encoding for two functional domains of malonyl-CoA reductase from C. aurantiacus (MCR-N, MCR-C), acetyl-CoA carboxylase from Y. lipolytica (ACC Y l ), and cytosolic NADH kinase from S. cerevisiae (cPOS S c ). The recombinant strain was fermented in a 5 L bioreactor by a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g·L −1 [ 20 ]. Yarrowia lipolytica , a representative unconventional yeast, is generally recognized as a safe strain (GRAS), and it can be safely used in supplements for people over 3 years old [ 21 ]. As an excellent heterologous expression chassis strain, Y. lipolytica synthesizes multiple metabolites, including organic acids, lipases, fatty acids, terpenoids, etc. [ 22 ]. Prof. Catherine Madzak modified Y. lipolytica (W29, ATCC20460), obtaining Po1d series strains (Po1d, Po1e, Po1f, Po1g, and Po1h), which knocked out the alkaline protease coding gene AEP and integrated gene SUC in the Po1d strain, which can cause the strain to use sucrose as substrate. Based on the Po1d strain, Po1f strain knocked out the acid protease coding gene AXP and eliminated the adverse effect of extracellular protease on the expression of the foreign protein [ 23 , 24 , 25 ]. In addition, Prof. Catherine Madzak also developed a series of pINA plasmids, including pINA1312 and pINA1269 [ 26 , 27 ]. The strong promoter hp4d was used in the pINA plasmids; it is a growth-dependent promoter that needs no induction and is scarcely affected by the environment, allowing the foreign protein to be expressed in the early stationary phase of cell growth [ 28 ]. To increase the efficiency of plasmid integration, the researchers added a zeta sequence to the plasmid. The function of the zeta sequence is that it improves the efficiency of foreign DNA integration into the genome of Y. lipolytica ( Ylt1 Δ) by non-homologous end joining (NHEJ) and provides a new way to integrate into the genome of Y. lipolytica by NHEJ [ 29 ]. In this study, the production of 3-HP using glucose as substrate was obtained in Y. lipolytica through the malonyl-CoA pathway ( Figure 1 A). We introduced a series of exogenous genes into this pathway, including MCR from C. aurantiacus , ACS L 641 P from Salmonella enterica , and overexpressed endogenous ACC1 . Meanwhile, the malonyl-CoA reductase was separated into two subunits, which showed higher 3-HP production. Further, GAPN from Streptococcus mutans was overexpressed to supply NADPH to the system, and the extra added pyruvic acid provided more precursors to the malonyl-CoA pathway. In addition, in order to ensure more fluxes to 3-HP, we knocked out genes MLS1 and CIT2 , which introduced more fluxes into the glyoxylate cycle. To solve resolve the 3-HP degradation, we also tried to break the malonate semialdehyde pathway in Y. lipolytica by knocking out genes HPDH and MMSDH , leading to greater accumulation of 3-HP ( Figure 1 B). Overall, the potential of Y. lipolytica as a promising chassis strain for 3-HP production was demonstrated for the first time. In comparison to prokaryotic chassis cells, Y. lipolytica has a more suitable fermentation environment for 3-HP (pKa: 4.5) and is more tolerant to the toxicity effects of 3-HP. Our study also provides new insights for increasing the production of 3-HP in the future.",
"discussion": "4. Discussion 3-HP plays an important role in industrial chemical production. Chemical synthesis methods have a series of disadvantages, including high cost, high pollutant emissions, and complex steps, and utilizing microorganisms to synthesize 3-HP is environmentally friendly. The increasing yield of the biosynthesis method is also advantageous. Prokaryotic chassis cells, such as E. coli and K. pneumoniae , and eukaryotic chassis cells, such as S. cerevisiae and P. pastoris , were used to produce 3-HP. E.coli , which was previously used for research, as the chassis cell has the advantages of high yield and easy operation. However, when synthesizing 3-HP, it has poor tolerance, which is a disadvantage. Y. lipolytica has a high tolerance for the production of organic acid products because its fermentation environment is acidic. Compared to S. cerevisiae , Y. lipolytica also has more metabolic flux for synthesizing 3-HP. As malonyl-CoA is the precursor for the synthesis of FFA [ 42 ], Y. lipolytica , a strain capable of synthesizing and storing large amounts of FFA, has a significant advantage in producing 3-HP . Therefore, we chose Y. lipolytica as the chassis cell to synthesize 3-HP in this study. As shown in Section 3.1 , the initial malonyl-CoA pathway for synthesizing 3-HP was constructed in Y. lipolytica . The yield of 3-HP reached 0.203 g·L −1 , which is higher than that of S. cerevisiae [ 17 ]. This also indicates that Y. lipolytica has a more abundant precursor than S. cerevisiae . In order to further enhance enzyme activity, according to the literature, MCR was split into two subunits: MCR-N and MCR-C. The yield of 3-HP reached 0.353 g·L −1 . The activity of MCR and replenishing of NADPH are the key to increasing the yield of 3-HP through the malonyl-CoA pathway [ 43 ]. Therefore, plasmid pINA1312 was used to connect their coding genes to perform multiple copies into genome of Y. lipolytica by NHEJ [ 44 ]. The results indicated that GAPN S m is more suitable for expression than GAPN B c as described in Section 3.2 . On this basis, a series of genes was overexpressed to promote the metabolic flux to flow towards malonyl-CoA. As shown in Section 3.3 , one overexpressed copy of ACC1 Y l and ACS S e L 641 P by plasmid pINA1269 can increase the yield of 3-HP to 0.745 g·L −1 . The results showed that transforming another two copies of the two genes into the genome of Y. lipolytica failed to improve the yield of 3-HP. It is inferred that the contents of acetate and acetyl-CoA in the strain are not the key factor limiting 3-HP production at this time. Likewise, the overexpression of the ALD6 gene cannot increase the production of 3-HP; this may be because Y. lipolytica is unable to perform aerobic alcohol fermentation, and the main metabolic flux automatically flows to acetate. This is not consistent with the situation in S. cerevisiae . In Section 3.4 , we used the CRISPR/Cas9 system to knock out genes related to the glyoxylate cycle, including MLS1 and CIT2 . The yield of 3-HP increased by 0.113 g·L −1 , which is higher than that of S. cerevisiae . By focusing on the production of 3-HP in the later stage of fermentation, we noticed the degradation of 3-HP. After querying the KEGG database, we discovered the presence of the malonate semialdehyde pathway, which can degrade 3-HP in Y. lipolytica . In Section 3.5 , the genes MMSDH and HPDH were knocked out to alleviate the degradation of 3-HP and increase the content of precursor for 3-HP synthesis. The yield of strain reached 1.128 g·L −1 , increasing by 0.269 g·L −1 , and significantly improved the production. Compared to the initial strain Po1f-tMCR, the yield of 3-HP improved 5.55-fold. This yield is higher than that of the recombinant S. cerevisiae strain achieved via shake flask culturing [ 45 ]. In Section 3.6 and Section 3.7 , a series of fermentation-level measures were implemented to increase the production of 3-HP. Adding 4 g·L −1 pyruvic acid to the YPD medium can further increase the production of 3-HP because pyruvic acid is also another carbon source. In the background of shake flask fermentation culture, the medium with a pH value of 6.0 is more suitable for the accumulation of 3-HP. OD 600 is a reflection of the growth status of the strain. The results showed that there is no significant difference in the OD 600 among different recombinant strains. This situation is consistent with the growth of recombinant strains of Y. lipolytica , which are used to produce other productions. To rule out the impact of 3-HP accumulation on strain growth, we tested the tolerance of Y. lipolytica to 3-HP. The results showed that the current 3-HP shake flask fermentation yield does not impact the growth of Y. lipolytica . Lastly, fed-batch fermentation was performed in a 1 L bioreactor to preliminarily verify the ability of strain Po1f-NC-14 to synthesize 3-HP in Section 3.8 . The accumulation concentration of 3-HP in the fermentation broth of Po1f-NC-14 reached 16.23 g·L −1 , which is higher than that in E. coli [ 11 ] and is close to that of an unconventional yeast P. pastoris [ 20 ]. Through the fed-batch fermentation results, it can also be observed that the OD 600 began to decrease and the speed rate of glucose consumption gradually slowed. As a result, the production of 3-HP no longer increased in the later stage of fermentation. It can be inferred that the volume of bioreactor is an important factor which limits strain growth and the yield of 3-HP. Although we employed a series of measures to increase the production of 3-HP, there are still many new directions to explore. Constructing a super yeast chassis can significantly improve the metabolic flux of precursor for synthesizing 3-HP, and related gene manipulations can be applied in Y. lipolytica [ 42 ]. We will also perform gene manipulation on malate dehydrogenase coding gene ( MDH ) and focus on the ration of two forms of MDH to further increase the yield of 3-HP. Attempting to apply differ expression promoters [ 46 ], setting up a dynamic sensor–regulator system to control the expression level of genes [ 47 ], and overexpressing relevant genes obtained from the results of RNA-sequencing of recombinant strain [ 48 ] can increase 3-HP production at the genetic level. At the same time, optimizing the fermentation conditions of fed-batch fermentation in the bioreactor can also improve the accumulation of 3-HP. Overall, using Y. lipolytica to produce and accumulate 3-HP has great potential for development."
} | 4,469 |
20431716 | null | s2 | 6,124 | {
"abstract": "The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO(2)) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO(2) into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction."
} | 400 |
35074913 | PMC8795527 | pmc | 6,125 | {
"abstract": "Significance We provide insights into the sequence–conformation–property relationship that is central to the mechanical properties of protein elastomers. We find that a high content of glycine residue alone, not including proline, is sufficient for achieving near-perfect resilience. The content of proline residue may be associated with the metastability of random coils. Also, Raman spectroscopy, as a potent tool for investigating the conformation–property relationship, gives rise to a direct correlation between semiquantitative Raman features and the magnitude of elastic resilience. Moreover, metastable conformation or conformational polymorphism is useful to develop continuously and mechanically graded protein materials that may exhibit unique structural merits. This work underlies the exploitation of natural and de novo–designed sequences for protein elastomers and materials.",
"discussion": "Results and Discussion We exploited the metastable random coils of regenerated silk fibroin to prove the concept of the proposed conformation-driven strategy ( Fig. 1 A and SI Appendix , Fig. S2 ). Before spinning, silk fibroin adopts predominantly random coils in native silk feedstocks because random coils help prevent premature protein aggregation during in vivo storage of the silk feedstock over weeks. After silk spinning, the predominant conformation of silk fibroin transforms from random coils to β-sheets ( Fig. 1 A and SI Appendix , Fig. S2 ) ( 32 , 33 ). The (meta)stability of silk fibroin conformations can be explained by the theory of the energy landscape ( 34 , 35 ), in which metastable random coils are assumed to be kinetically stabilized in a local energy minimum, in contrast to the stable β-sheets exhibiting the lowest global energy ( Fig. 1 A ). Accordingly, the random coil to β-sheet transition is largely inevitable for silk fibroin, unlike naturally occurring resilient proteins that adopt stable random coil structures ( 2 , 28 ). Notably, the conformational transition of silk fibroin can be impeded and accelerated by hyaluronic acid ( 20 ) and methanol ( 36 ), respectively, as examples of many options ( 23 , 32 ). The kinetical control of conformations is likely based on tuning energy barriers or pathways between local and global minima. The characteristic sequences of silk fibroin are associated with the metastable conformation and material properties such as resilience and hydrogel hydration. Silk fibroin (heavy chain) is composed of alternately positioned hydrophilic and hydrophobic domains ( Fig. 1 B ). The hydrophilic domains contain hydrophilic and negatively charged amino acids, such as aspartic acid (D), glutamic acid (E), and serine (S), as well as a characteristic 25-residue sequence motif, SGFGPYVANGGYSGYEYAWSSESDF. The hydrophobic domain contains primarily glycine (G), alanine (A), and S, as well as motifs, such as GAGAS and GAAS. Notably, hydrophobic domains contain no charged residues. Thus, the hydration of silk fibroin hydrogels is primarily attributed to hydrophilic domains. In addition, the motif GAGAS in the hydrophobic domain can adopt either random coils or β-sheets, allowing the metastable conformation of silk fibroin. In contrast, the hydrophilic domain lacks specific motifs for regular secondary structure and thus largely remains as random coils. Comparing amino acids between silk fibroin and naturally occurring resilient proteins provides insights into the molecular mechanism of resilience. The G content of silk fibroin is around 45.9 mol%, which is comparable to other resilient proteins such as aortic elastin (30 mol%) and insect resilin (39 to 42 mol%) ( SI Appendix , Table S1 ). G is hydrophobic and has the smallest side chain, a hydrogen atom, thus benefiting chain flexibility and entropy elasticity of protein materials ( 10 , 37 ). In addition, the proline content of silk fibroin (P, 0.3 mol%) is much lower than that found in naturally occurring resilient proteins such as elastin (P, 12 mol%) and resilin (P, 7 to 12 mol%) ( SI Appendix , Table S1 ) ( 38 ). Proline has a unique five-member ring with restricted backbone conformations and has been known to resist the formation of β-sheets ( 38 ). The low proline content of silk fibroin may explain, in part, the capability to form β-sheets. Thus, we suggested that a high glycine content seems sufficient for the exceptional resilience and that the low proline content would not compromise the magnitude of the elastic resilience but seems associated with the metastable random coil conformation. We cross-linked regenerated silk fibroin in solution to form hydrogels via a ruthenium [Ru(II)]/persulfate–mediated approach that is based on the photo-oxidation and coupling of tyrosine residues ( 39 – 41 ) ( Fig. 1 C ). The dynamic photo–cross-linking process was investigated by three measurements: dityrosine fluorescence, damping factor [tan(δ)], and water content ( Fig. 2 A and D and SI Appendix , Figs. S4–S6 and Supplementary Text ). We also investigated cell encapsulation and in vitro degradation to imply the biomedical potential of photo–cross-linked silk fibroin hydrogels ( SI Appendix , Fig. S7 and Supplementary Text ). C3H/10T1/2s cells, encapsulated in the silk fibroin hydrogels, exhibited cytoskeleton development and considerable viability and metabolic activity. Fig. 2. Conformational characterizations of silk fibroin materials. ( A ) Fluorescence excitation-emission matrix spectra at crosslinking times from 0 to 180 s. Characteristic fluorescence of dityrosine crosslinks and autofluorescence of silk fibroin and Ru(II) are indicated. ( B ) Raman spectra at crosslinking times from 0 to 180 s, compared with rigid hydrogels. Two regions, including amide I band (at around 1,667 cm −1 ) and tyrosine doublet (Tyr, at 850 and 830 cm −1 ), are labeled. ( C ) FTIR amide I band of silk fibroin solutions, swollen, and rigid hydrogels. Deconvolution of the FTIR amide I band gives rise to semiquantitative content of three major conformations/secondary structures of silk fibroin, including random coils, β-sheets, and β-turns. ( D ) Random coil–dominated conformations remained after photo–cross-linking, as evidenced by dityrosine fluorescence and FWHM of Raman amide I band. ( E and F ) FWHM of Raman amide I band and Tyr ratio (I 850 /I 830 ) of silk fibroin solutions, as-prepared, and rigid hydrogels. ( G ) Conformation content estimated by deconvoluted FTIR amide I band. ( n = 3 independent spectra, **** P < 0.0001, * P < 0.1, n.s. P > 0.1, single-factor ANOVA followed by Scheffé's post hoc test). We prepared a set of silk fibroin hydrogels with distinct conformations and mechanical properties, including “as-prepared,” “swollen,” and “rigid,” which provides insights into the conformation-based resilience on a comparative basis ( Fig. 1 D ). The silk hydrogel, after photo–cross-linking, is termed as-prepared. It is not treated with solvents for swelling, thus maintaining the same water content and chemical composition (including unreacted cross-linking reagents) as the photo–cross-linking precursor solution. The “swollen hydrogel is obtained by incubating as-prepared ones in 0.9 wt/vol% sodium chloride for 3 d ( SI Appendix , Figs. S2 and S6 ). The incubation swells the hydrogel and washes away the unreacted reagents and polypeptide chains. As a result, water content increases from 75% (as-prepared) to over 85% (swollen) ( SI Appendix , Fig. S6 ); the color of the silk hydrogel changes from orange (as-prepared) to light yellow (swollen) due to the removal of colored reagents such as Ru(II) ( SI Appendix , Fig. S6 ). The aqueous solution of 25 wt% silk fibroin is also in light yellow. The rigid hydrogel is obtained by incubating swollen ones in 90 vol/vol% methanol for 1 h, transforming random coils into β-sheets ( SI Appendix , Fig. S2 ). The name “rigid” is coined out of the mechanical property; the rigid hydrogel exhibits an increased Young’s modulus and decreased resilience compared to the other two hydrogels ( Fig. 1 D and SI Appendix , Fig. S9 ). The rigid hydrogel is immersed in 1× phosphate-buffered saline (PBS) and remains hydrated throughout this work. Raman and Fourier-transform infrared (FTIR) spectroscopies were utilized to characterize the conformation of silk fibroin materials ( Fig. 2 and SI Appendix , Fig. S8 ). The conformation-specific results of both Raman and FITR spectra have been verified by nuclear magnetic resonance (NMR), circular dichroism, and wide/small-angle X-ray scattering (WAXS/SAXS) ( 29 , 42 ). Suggested by the spectral results, silk fibroin solution, as-prepared, and swollen hydrogels were dominated by random coils, in contrast to β-sheet–dominated rigid hydrogels ( Fig. 2 and SI Appendix , Fig. S8 and Supplementary Text ). Random coil–related characteristics of Raman spectra include amide I band (C = O stretching) at 1,667 cm −1 , tyrosine doublet at 850 cm −1 and 830 cm −1 (Tyr), amide III band (mainly C-N stretching) at 1,251 cm −1 , and two backbone stretching vibrations (C-C) at 1,103 cm −1 and 942 cm −1 ( 43 , 44 ). In particular, the full width at half maximum (FWHM) of Raman amide I and the Raman Tyr ratio (I 850 /I 830 ) were used for semiquantitative comparisons of protein conformations ( 42 , 45 ). For solutions and as-prepared and swollen hydrogels, the FWHM of amide I and the Tyr ratio remained around 59 cm −1 and above 3, respectively, suggesting that random coil–dominated conformations largely remained after cross-linking and swelling ( Fig. 2 D – F and SI Appendix , Fig. S8 ). The dityrosine crosslink likely restricts the vibration of tyrosine phenol rings, thus resulting in the decreased Raman Tyr ratio from 3.6 ± 0.1 to 3.3 ± 0.2; the swelling of the molecular networks may facilitate the ring vibration, increasing the Tyr ratio to 4.1 ± 0.6. We also used deconvoluted FTIR spectra ( 46 ) to corroborate the random coil–dominated conformations of silk fibroin solutions and swollen hydrogels ( Fig. 2 C and G ). The solution and the swollen hydrogel exhibit the FTIR amide I band at 1,647 cm −1 and 1,645 cm −1 , respectively, indicating random coil dominance. In contrast, the FTIR amide I of the rigid hydrogel at 1,621 cm −1 indicates β-sheet dominance. Also, FTIR deconvolution estimated the random coil content of the solution and the swollen hydrogel, which is 78 ± 5% and 77 ± 7%, respectively, compared with the low content of β-sheets (2%), thus verifying the random coil dominance ( Fig. 2 G ). Notably, several protein hydrogels exhibited conformational changes after cross-linking, such as enzyme–cross-linked silk fibroin elastomers ( 19 ). The improvement in the current work may be attributed to the short cross-linking time of the Ru(II)/persulfate approach (2 to 3 min) compared with that of the enzymatic approach (30 to 60 min); short cross-linking time minimizes the disturbance to the initial conformation ( 39 , 40 ). The random coil–dominated conformation is a prerequisite for the entropy elasticity of protein hydrogels. We employed uniaxial tensile tests and dynamic mechanical analysis (DMA) to characterize the elastic resilience of the three silk fibroin hydrogels, including as-prepared, swollen, and rigid ( Fig. 3 , SI Appendix , Figs. S9–S11 , and Movie S1 ). At 0.1 mm/mm tensile strain, as-prepared and swollen hydrogels exhibited near-perfect resilience of 98.2 ± 0.7% and 97.3 ± 1.3%, respectively, in contrast to rigid hydrogels with a relatively low resilience of 86.2 ± 0.9% ( Fig. 3 B and C and SI Appendix , Fig. S9 ). The resilience of as-prepared hydrogels remained stable at 96.8 ± 1.1% with increased tensile strains from 0.1 mm/mm to 0.9 mm/mm ( Fig. 3 B and C ) and at 97.5 ± 0.5% with 20 consecutive stretch–recoil cycles and 0.5 mm/mm strain ( SI Appendix , Fig. S9 ). Swollen hydrogels maintained largely above 95% resilience at increased tensile strains up to 0.6 mm/mm ( Fig. 3 C and SI Appendix , Fig. S9 ). In contrast, the resilience of rigid hydrogels exhibited a significant reduction from 86.2 ± 0.9% at 0.1 mm/mm to 70.8 ± 4.4% at 0.2 mm/mm ( SI Appendix , Fig. S9 ). Similarly, a globular protein-based elastomer (GRG 5 RG 4 R) exhibited a drastic strain-dependent decrease in resilience from near 100% at 0.1 mm/mm to around 76% at 0.9 mm/mm ( 4 ). The reduced resilience was due to the energy-dissipated unfolding of force-resistant globular domains ( 4 ) and β-sheets ( 35 ). In addition, DMA results verified the exceptional resilience of as-prepared and swollen hydrogels, which, at 1 Hz, are 95% and 91%, respectively ( Fig. 3 E and SI Appendix , Fig. S10 ). In contrast, the resilience of rigid hydrogels is 77% at 1 Hz. We also discussed other mechanical differences between silk fibroin hydrogels, associated with the swelling of the molecular network and the conformational transformation, in SI Appendix , Supplementary Text . Fig. 3. Mechanical characterizations of silk fibroin hydrogels. ( A ) Optical images of as-prepared hydrogels stretched over 1 mm/mm strain in uniaxial tensile tests. ( B ) Consecutive cyclic tensile tests of as-prepared hydrogels with increased final strains from 0.1 to 1 mm/mm. The curves are shifted along the x-axis for clarity. Inset shows superimposable curves, indicating hydrogel recoverability. Red dashed curves indicate fitting results with statistical rubber theory and begin to deviate roughly at 0.5 mm/mm strain. ( C ) Resilience and tensile modulus of both as-prepared and swollen hydrogels as a function of final strains. ( D ) Comparison of silk fibroin hydrogels to other resilient protein materials in terms of resilience and tensile stress, also provided in SI Appendix , Table S2 . Inset highlights data points within the red dashed box. ( E ) Resilience of silk fibroin hydrogels tested by DMA, in comparison to replotted data of resilin and elastin from ref. 10 . ( F ) Direct correlation between elastic resilience and conformation-specific Raman features, including Raman Tyr ratio (I 850 /I 830 ) and FWHM of the Raman amide I band. Methanol treatment from 0 to 3 min was used to induce incremental change of conformations and mechanical properties. Red dashed lines indicate linear regressions with corresponding Pearson’s correlation coefficients, r . The resilience of as-prepared and swollen silk fibroin hydrogels was superior to or comparable with the most resilient protein elastomers known, including dragonfly tendon resilin (92%), bovine ligament elastin (90%), tendon collagen (90%), abductin (96%), recombinant resilin/resilin-like proteins (up to 97%), ELP (up to 84%), and methacryloyl-modified tropoelastin (76%) ( Fig. 3 D and SI Appendix , Table S2 ). Also, the DMA-characterized resilience of as-prepared and swollen silk fibroin hydrogels was almost coincident with resilin and elastin in the overlapped range of frequency roughly from 1 to 10 Hz ( Fig. 3 E ). The resilience of as-prepared and swollen hydrogels was also superior to natural silk materials (around 30%) and other silk fibroin hydrogels (up to 94.6% by compression tests) ( SI Appendix , Tables S2 and S3 ). The improved resilience was primarily due to the well-maintained predominance of random coils, in contrast to β-sheet dominance of natural silk materials ( Fig. 1 A ) ( 32 , 35 ). These results highlighted the feasibility of random coil–forming non-resilin/elastin sequences, such as that of silk fibroin, to achieve the resilience as exceptional as that of resilin and elastin, thus providing avenues for developing resilient protein materials. We examined the conformational origin of the elastic resilience of silk fibroin hydrogels by fitting the tensile stress–strain curves with the statistical rubber theory ( Fig. 3 B and SI Appendix , Fig. S9 ) ( 10 ). The as-prepared and swollen hydrogels agreed well with the statistical rubber theory below 0.5 and 0.3 mm/mm strains, respectively, verifying the rubber-like/random coil nature of as-prepared and swollen hydrogels. Notably, the fitting range was wider than the 0.25-mm/mm strain of a recombinant resilin protein ( 13 ). We ascribed this difference to the higher molecular weight of the regenerated silk fibroin (around 100 kDa) than the resilin protein (28.5 kDa), because longer molecular chains may lead to more random links, thus better fitting the Gaussian assumption of the rubber network theory ( 10 ). Also, the higher molecular weight may be advantageous for ultimate tensile strength and extensibility ( SI Appendix , Table S2 and Supplementary Text ). Another notable finding of this study is the direct correlation between elastic resilience and Raman-characterized conformations, supplementing conventional conformation–property relationships of protein materials. We treated swollen silk fibroin hydrogels with 90 vol/vol% methanol for 0, 1, 2, or 3 min ( Fig. 3 F and SI Appendix , Fig. S11 ). With increased treatment times, the Raman Tyr ratio and the FWHM of Raman amide I decreased from 3.3 ± 0.2 to 1.9 ± 0.1 and from 58.6 ± 2.1 cm −1 to 28.7 ± 1.4 cm −1 , respectively, implying the incremental transition from random coils to β-sheets. Also, the resilience decreased from 97.3 ± 1.3% to 65.7 ± 2.9%, and Young’s modulus increased from 67.7 ± 1.6 kPa to 1.2 ± 0.4 MPa ( Fig. 2 B and SI Appendix , Fig. S9 ). Furthermore, the elastic resilience was directly correlated to the Raman Tyr ratio and the FWHM with Pearson’s correlation coefficients of 0.97 and 0.94, respectively ( Fig. 3 F ). These results indicated an early part of the transforming process from swollen to rigid hydrogels, supported the conformational origin of the elastic resilience, and suggested the potential to modulate resilience via tuning conformations. Also, due to the direct correlation, Raman spectroscopy may be a promising tool for exploring conformation-based elasticity. We further exploited the conformation-driven strategy and the metastable random coils of silk fibroin to construct mechanically graded protein materials ( Fig. 4 and SI Appendix , Figs. S12–S15 ). Protein conformations have been used to tune the modulus and strength of individual materials ( 22 , 23 ) yet failed to produce continuously graded materials that are ubiquitous in living organisms ( 35 , 47 ) and allow unique structural merits ( 48 ). Also, protein conformation may be a viable alternative to the material composition found in naturally occurring graded protein tissues ( 35 , 47 ) ( SI Appendix , Table S4 ) for making graded materials. Protein conformation can be dynamically controlled ( 24 , 25 ) and thus leads to a dynamic mechanical gradient. Fig. 4. Graded protein materials. ( A ) Schematics of directional methanol treatment and resultant graded silk hydrogels with spatially controlled conformations and mechanical properties. Graded hydrogels are roughly composed of a resilient end, a rigid end, and a middle transition zone. ( B ) Optical and fluorescent images of graded hydrogels. Blue fluorescence at 300 nm excitation is the characteristic of dityrosine cross-links. ( C ) Mapping spectra of Raman amide I band and Raman tyrosine doublet (Tyr, at 850 cm −1 and 830 cm −1 ) over around 2.5 mm in the transition region. Arrows indicate the direction of resilient and rigid ends. ( D ) Typical cyclic compression curves of cylindrical samples punched out from local regions of graded hydrogels. ( E ) Simulated and experimental results of swollen and graded silk hydrogels after 180° rotation. Straight arrows indicate the center of rotating spirals. The ends of graded hydrogels are fixed under glass slides. We developed a directional methanol-treatment method to realize the spatial gradient of protein conformation ( Fig. 4 and SI Appendix , Fig. S12 and Supplementary Text ). Raman characterization suggested random coil–dominated and β-sheet–dominated conformations at the two ends of graded hydrogels, respectively ( SI Appendix , Fig. S13 ). In the middle transition region, the FWHM of Raman amide I and the Raman Tyr ratio spatially changed from 55 cm −1 to 28 cm −1 and from 3.8 to 2.4, respectively, implying a continuous spatial transformation from random coils to β-sheets ( Fig. 4 C and SI Appendix , Fig. S13 ). Also, we used cyclic compression tests to characterize the local mechanical properties of graded hydrogels ( Fig. 4 D and SI Appendix , Fig. S14 ). The resilient end of the graded hydrogel exhibited 55.5 ± 1.5 kPa modulus and 92.8 ± 3.4% resilience; the rigid end exhibited 732.1 ± 19.5 kPa and 53.6 ± 1.2%; and the transition zone exhibited 149.1 ± 11.7 kPa and 68.9 ± 4.1% in the middle between that of the two ends due to the transitioning nature. Furthermore, we developed a finite element-based model of the graded silk fibroin hydrogels, based on the actual size and the experimentally obtained moduli ( Fig. 4 E and SI Appendix , Fig. S15 ). Simulation results of graded hydrogels with 180° rotation agreed well with experimental results, including the rotated shape and the spiral position. These results corroborated the graded silk fibroin hydrogels with both conformational and mechanical gradients and verified the conformation-driven strategy for making graded protein material. In conclusion, we exploited protein conformation of non-resilin/elastin sequences to construct exceptionally resilient and graded protein hydrogels. The direct correlation between conformation-related Raman features and elastic resilience provides a semiquantitative tool to probe the molecular mechanism of protein elastomers. The conformation-driven strategy would be generally useful for exploiting naturally occurring sequences and de novo – designed variants to develop a range of elastic protein biomaterials."
} | 5,451 |
38324676 | PMC10849601 | pmc | 6,126 | {
"abstract": "Through evolution, nature has presented a set of remarkable protein materials, including elastins, silks, keratins and collagens with superior mechanical performances that play crucial roles in mechanobiology. However, going beyond natural designs to discover proteins that meet specified mechanical properties remains challenging. Here, we report a generative model that predicts protein designs to meet complex nonlinear mechanical property-design objectives. Our model leverages deep knowledge on protein sequences from a pretrained protein language model and maps mechanical unfolding responses to create proteins. Via full-atom molecular simulations for direct validation, we demonstrate that the designed proteins are de novo, and fulfill the targeted mechanical properties, including unfolding energy and mechanical strength, as well as the detailed unfolding force-separation curves. Our model offers rapid pathways to explore the enormous mechanobiological protein sequence space unconstrained by biological synthesis, using mechanical features as the target to enable the discovery of protein materials with superior mechanical properties.",
"introduction": "INTRODUCTION Proteins present an elegant yet complex and rich design platform. The various functions and outstanding properties of proteins can be attributed to the folded three-dimensional (3D) structures, encoded by the underlying one-dimensional (1D) primary sequences consisting of about 20 naturally occurring amino acids ( 1 ). Through evolution, nature has demonstrated great success in “designing” proteins as a set of critical building blocks that constitute fundamental functions of all life, and specifically remarkable biomaterials, ranging from the structural hierarchies in collagens, complex assemblies such as silk, to tissue assemblies such as muscle and skin ( 2 – 5 ). In these various tissues and systems, the detailed mechanical signature—often, their response to mechanical pulling—is an essential feature for mechanobiology ( 6 – 9 ). At the same time, there remains vast design space of mechanically optimized proteins yet unexplored by nature given the enormous possibilities of protein sequences ( 10 ). Hence, inspired by nature, discovering de novo proteins may unlock potentially unprecedented properties and functions ( 3 , 10 – 16 ). However, this enormous design space and costs associated with experimental testing present great challenges in finding effective tools to design de novo protein sequences that meet a set of interested functions or properties ( 14 – 19 ). In recent years, the development of deep learning approaches and their applications to proteins have provided fast avenues for protein study and design. For forward problems focused on structure identification, deep learning–based tools such as AlphaFold2 ( 20 ) and RoseTTAFold ( 21 ) represent a breakthrough in achieving competitive accuracy with experimental methods in predicting 3D folded structures based on protein sequences at a much reduced cost. ( 22 ) Built upon these approaches, other protein folding tools [e.g., Omegafold ( 23 ), RGN2 ( 24 ), HelixFold-single ( 25 ), and ESMFold ( 26 )] have been exploring the application of large language models. By removing dependence on multiple sequence alignments (MSAs) as the input, improvements in further reducing computational costs and achieving better predictions for orphan and rapidly evolving proteins have been demonstrated ( 23 , 24 , 26 , 27 ). End-to-end models based on deep learning that predict various structural features [e.g., secondary structure type and content ( 28 – 33 ), binding sites ( 34 ), and surfaces ( 35 )] and properties [e.g., solubility ( 16 , 36 , 37 ), melting temperature ( 38 ), natural vibrational frequencies ( 39 , 40 ), and strength ( 41 )] for given sequences have also been reported. At the sample time, the inverse design of de novo proteins that meet desired structural or property features presents a more challenging task. On one hand, facing the enormous sequence space, search algorithms teamed with efficient deep learning–based forward predictors ( 30 , 42 , 43 ) may still suffer from inefficient exploration and the design accuracy and varieties of the discovered sequences are not easily controlled. On the other hand, recently emergent generative models ( 44 – 49 ) provide a direct map from the desired characteristics to potential designs and are becoming an emerging paradigm for various materials research and design ( 50 – 55 ), including proteins. For example, using an attention-based diffusion model trained on secondary structure data, de novo protein sequences can be generated based on secondary structure design objectives ( 56 ). However, these generative design models often focus on structural level design [such as secondary structures ( 56 ) or detailed protein backbone shapes ( 57 – 60 )]. In contrast, development of generative models aimed at end-to-end design from property of interest to protein sequence remains rare ( 61 ). Here, we focus on nanomechanical properties ( 62 – 65 ) of proteins. Thanks to the advent of single-molecule technology ( 66 ) [e.g., atomic force microscopy (AFM) ( 67 , 68 ), optical tweezers ( 69 , 70 ), and magnetic tweezers ( 71 , 72 )], the measurement of protein unfolding under an applied mechanical force provides a unique molecular basis for understanding protein deformation (elasticity/plasticity) and fracture ( 64 ) and can play key roles in affecting some macroscopic mechanical properties of protein-based materials due to the inherent structural hierarchy. For example, via experimental measurements and theoretical analysis, it has been demonstrated that toughness of synthetic protein hydrogels can be correlated to the mechanical unfolding responses of the protein molecules and that mechanically strong folded proteins can result in tough hydrogel designs ( 73 ). Therefore, generating de novo proteins that meet desired mechanical unfolding responses can represent a key molecular level design step in protein-based material designs. Compared with previous protein design cases, this problem presents some unique challenges. First, this is a property-to-sequence end-to-end design task bypassing the structure level, which is expected to be more difficult than previous structure-to-sequence design tasks ( 56 , 60 ). Second, the available or affordable data on mechanical unfolding responses of known proteins ( 74 ) are rare when compared to those for protein structures ( 75 ) or sequences ( 76 ). Besides mechanical properties, we expect that these two challenges are also shared by many other property-to-sequence design tasks in proteins. To address this problem, here, we combine an attention-based ( 77 ) diffusion model ( 56 ) with a pretrained large language model ( 26 ) for proteins to construct a generative deep learning model that predicts amino acid sequences and 3D protein structures based on mechanical unfolding responses as design objectives. In a singular workflow ( Fig. 1 ), we start with performing a large series of full-atom molecular dynamics (MD) to simulate the mechanical unfolding process of Protein Data Bank (PDB) ( 75 ) proteins and recording the force responses ( Fig. 1A ). Then, we construct a protein language diffusion model (pLDM) by translating the protein sequences into a word probability latent space using a pretrained protein language model (pLM) and training a diffusion model to learn the map between sequence representations and the force-separation responses ( Fig. 1B ). At deployment, the trained pLDM predicts sequence candidates based on the given unfolding force conditions and the integrated folding algorithm (i.e., OmageFold) ( 23 ) determines the 3D structures of the resulting sequences. For validation, we compare the designed sequences with known proteins to analyze novelty ( Fig. 1C ) and test the designed proteins using MD to compare the mechanical properties and unfolding responses with input conditions. To prepare the design pipeline for further experimental validation, other properties key to experimental synthesis and testing, such as solvent accessible surface area (SASA) ( 78 ), solubility, or stability ( 36 , \n 79 ), can be estimated using available predicting tools ( 36 , \n 78 , \n 79 ) to further screen for preferred protein candidates ( Fig. 1D ). Through well-controlled comparisons, we demonstrate that our pLDM outperforms the vanilla diffusion model with or without an iterative design scheme. Built upon the property-to-sequence generation capability of our model and the broad potential of protein materials in achieving superior mechanical properties, as well as other interesting properties ( 80 – 83 ) [e.g., optical ( 82 , \n 83 ), electronic ( 80 ), energy storage ( 81 ), etc.], we expect that our end-to-end design model can be useful in numerous biological and engineering applications for the property-targeted generative design of various protein material systems. Fig. 1. Workflow of developing the end-to-end protein generation model. ( A ) Curating a PDB protein dataset on their mechanical properties by unfolding protein chains by force in MD simulations. ( B ) Overview of the conditioned protein language diffusion model (pLDM) developed here. ( C ) Analyzing the novelty of the generated protein sequences via protein-protein BLAST tests. ( D ) Validating the mechanical properties of the designed protein candidates using folding tools and mechanical unfolding tests and predicting other properties (e.g., solubility or stability) for further screening of the desired protein candidates.",
"discussion": "DISCUSSION Generating de novo proteins based on their mechanical unfolding responses presents unique challenges in property-targeted protein design. For rational design strategies, it is hard to grasp the complex relationships between sequences, structures, and properties. For data-driven methods, the labeled data on the mechanical properties are often costly to collect and limited in number, especially given the enormous possibilities in protein sequence space. Here, we have developed a pLDM as an effective tool to tackle these challenges and generate de novo proteins that meet the mechanical properties’ design objectives in an end-to-end manner. The pLDM developed combines the pretrained pLM and diffusion model as key components and leverages the strength of both. The pLM part is pretrained on the abundant protein sequence data and thus provides an effective representation of protein sequences in its latent space. The diffusion model part only operates in this latent space and learns the map between detailed pulling force responses and the sequence representation using only a relatively small set of data curated by performing full-atom simulations. By examining the unfolding details of individual designs and the statistics of the mechanical properties of many cases, we demonstrate that the proteins designed by our model meet the targeted overall mechanical properties, including toughness and strength, as well as the detailed unfolding force vectors with reasonably good accuracy. Moreover, the sequences generated are mostly de novo, sharing very limited similarity with existing/known proteins. Given the mechanical unfolding responses from known PDB proteins as the design input, our model still shows a strong tendency in discovering de novo proteins as alternatives. Constructing de novo unfolding responses as the input via a mixing scheme further boosts the probability of generating de novo designs. Finally, through controlled comparisons, we show that the pLDM outperforms the vanilla pDM with or without an iterative design scheme in achieving better design accuracy, thus clearly demonstrating the benefits of combining pretrained pLM and diffusion model in the pLDM developed here. A short summary of these key aspects about the pLDM is listed in Table 4 . Table 4. A short summary of the performance of protein language diffusion model developed in the present work and other models discussed. Model name Tested input conditions Design accuracy Design novelty The developed model: protein language diffusion model using one-shot design Mechanical unfolding responses from naturally existing proteins Good agreement with the designed pulling force responses as well as the strength and toughness in trend and values Tend to generate de novo ones, but can also rediscover ones that show some similarity to existing proteins De novo mechanical unfolding responses Similar to the above More probable to discover de novo sequences AM1: Protein diffusion model using one-shot design Mechanical unfolding responses from existing proteins Slightly weaker than AM2 – AM2: Protein diffusion model using multi-shot iterative design Mechanical unfolding responses from existing proteins Weaker than the developed model – As the initial steps of developing property-to-sequence generative models for de novo protein design, here we adopt the force-separation curves collected from MD simulations, in the hope of achieving consistency and relevance, avoiding bias from simplified models, and curating sufficient data points for DL model training. A few clarifications on the mechanical unfolding response data are included in the following. First, there exist differences as well as commonalities between MD results and experimental measurements of the force-separation curves of protein unfolding that deserve more nuanced discussion. The mechanical unfolding process of proteins often involves entropic elasticity, a transition to energetic elasticity, and bond breaking ( 95 ). Thus, the force response often shows strong rate dependence. Limited by computational power, the MD simulations are performed at a pulling speed several orders faster than that in the experimental tests. Therefore, the corresponding unfolding mechanisms (e.g., sequential or simultaneous rupture of several hydrogen bonds) can be different ( 96 ), and a direct comparison of the force records is often challenging. It should be pointed out that our current model, trained with all-atom MD data, is not intended for designing proteins that directly meet the given pulling force response at a different pulling speed. Instead, we use the MD data under the fixed pulling speed as a consistent representation of mechanical properties of protein. While the absolute values of strength and toughness measured by MD may change with the pulling speed, the relative rank of the mechanical properties of the proteins often remain robust. At the same time, there do exist methods and procedures to bridge MD and experimental results ( 97 , \n 98 ). For example, built upon the steered MD trajectory calculated here, further MD simulations can be performed to calculate the mean force potential during unfolding using statistical sampling methods. Unfolding force distribution in experimentally relevant regimes can be predicted based on the mean force potential via transition-state theory and Monte Carlo simulations ( 97 ). Therefore, the design goal of our current model can be connected to the response under experimental relevant pulling speed and force level with extra calculations and sampling efforts. Second, our MD data include fundamental atomistic details and avoid bias from bottom-up coarse-grained (CG) or theoretical models with specific assumptions. By tracking atomic motions during unfolding, the MD results require little predefined assumptions like those in CG models ( 99 ) or theoretical worm-like chain (WLC) models ( 100 ). At the same time, the information collected from the full-atom MD simulation can be used to fit parameters in CG and WLC models. The diffusion model was directly trained on these force patterns and learned to pick relevant features via the attention mechanisms embedded, avoiding any human intervention or pre-knowledge on the subject. Third, similar models can be developed when sufficient data on mechanical unfolding force under other testing protocols become available. As an initial step to prove this framework, our current model is trained and validated with the freshly curated MD data and the consistent MD test protocol. At the same time, similar models can be straightforwardly trained with other sufficient databases. In particular, when a large number of force-separation curves measured from a standard experimental protocol become available, models with similar architectures can be developed via direct training or transfer learning with them. With such models, consistent validation will require synthesis and test of the designed proteins in the wet lab. This requires well-planned in-depth design for specific goals while the current work is focused on model development and has provided self-consistent validation. We will save the experimental studies for future study. Finally, the specific choice of pulling force direction in our MD protocol is clarified here. It has been demonstrated that the mechanical unfolding responses of proteins can be affected by the detailed folding geometry and unfolding pathway ( 101 ). To effectively record the force history that corresponds to the deformation and uncoiling events of protein internal structures, we designed the test protocol to apply the mechanical force along the direction that connects the two ends of a protein chain after relaxation. When the mechanical pull is applied in another direction, the monomer is likely to first undergo a rotational motion to align along with the current pull direction before meaningful unfolding events happen given that in a quasi-static loading process rotation usually requires a smaller load than unfolding events like breaking hydrogen bonds. Once the protein monomer aligns along with the pulling direction, for the unfolding process that follows, we expect that a force record pattern similar to ours could be collected and our protocol and results remain relevant and robust. At the same time, some extreme cases could exist. For protein monomers mainly with weak internal folding structures (e.g., unstructured random coils), the load to rotate the monomer can be comparable to or larger than that of deformation and unfolding. We expect the detailed force pattern could be affected more strongly by the choice of pull direction in those cases. The freshly curated MD dataset and the pLDM developed here offer a unique and powerful means to investigate the underlying sequence-structure-property relationship and explore the enormous protein sequence spaces with molecular mechanical properties as guidance, to meet specific mechanobiology properties. With the available dataset, one can conduct a detailed survey on the internal structural features (e.g., secondary structures) of the proteins and their correlation with the unfolding force pattern to see whether there is any uneven distribution centered on certain secondary structure patterns (e.g., α helix and β sheet) among PDB proteins and our training set for certain unfolding force patterns. Applying our model, future studies can start with a systematic study on the relationships between sequence-structure-mechanical properties in proteins. For example, as demonstrated in Fig. 6 , one can construct de novo mechanical unfolding responses by mixing those existing PDB proteins at different ratios and our model can generate protein candidates that meet those mechanical unfolding responses. With such generating capability, one can systematically classify the patterns of various unfolding responses of the existing PDB proteins as shown in Fig. 2C , construct de novo force-separation responses transferring between those different patterns, and generate the corresponding proteins, thus studying how the patterns of the unfolding force responses and their transition affect the protein sequence mutations and internal structure variations. At the same time, as the designed de novo proteins increase in number, their sequences and mechanical unfolding responses can be used as growing data to gradually increase the protein dataset on mechanical properties and our model can be further trained on this growing set. With the more powerful model, one can further study some challenging topics, such as designing proteins with optimal mechanical properties or even their combination in various engineering and biological applications. While we have developed the pLDM that takes the mechanical unfolding responses as the design conditions here, we expect that similar pLDM frameworks can be generalized for other property-to-sequence design tasks in proteins. The enhancement brought by merging pretrained pLMs can be inspiring for other design tasks, especially where only small datasets on the property of interest are available or affordable at the beginning. At the same time, going beyond only one type of condition as the design target, our pLDM can also be generalized for design tasks under multiple objectives, given the flexibility of the diffusion model in incorporating these conditions ( Fig. 3B ). Combining the previous work using a pDM ( 56 ), one example can be taking both secondary structure and unfolding forces as the design target. Also, during the generation process, techniques like inpainting through selective masking or biasing certain amino acids ( 102 ) are straightforward to implement. Combining these under the pLDM framework, we envision a comprehensive generative model that moves towards designing proteins at all levels, including sequence, structure, and properties in harmony."
} | 5,397 |
29802781 | PMC6334530 | pmc | 6,129 | {
"abstract": "Summary Bacterial biofilms are communities of microbial cells encased within a self‐produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibres of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis , whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid‐like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross‐complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.",
"introduction": "Introduction Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling (Costerton et al ., 1987 ). The microbial cells resident in the biofilm are encased within a self‐produced extracellular polymeric matrix that commonly comprises lipids, proteins, extracellular DNA and exopolysaccharides (Flemming and Wingender, 2010 ; Hobley et al ., 2015 ). This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment, to supporting signal transduction and nutrient adsorption (Flemming and Wingender, 2010 ; Dragoš and Kovács, 2017 ; Vidakovic et al ., 2018 ). Bacillus subtilis is a soil dwelling bacterium that is a model for biofilm formation by Gram‐positive bacteria; beyond this it is of commercial interest due to its biocontrol and plant growth promoting properties that highlight its potential to substitute for petrochemical derived pesticides and fertilizers (Bais et al ., 2004 ; Chen et al ., 2012 , 2013 ). Biofilm formation is subject to complex regulatory pathways (Cairns et al ., 2014 ) and it is known that the B. subtilis biofilm matrix predominantly comprises three specific components. The first is an exopolysaccharide that serves to retain moisture within the biofilm and functions as a signalling molecule (Seminara et al ., 2012 ; Elsholz et al ., 2014 ). The composition of the exopolysaccharide remains unclear due to three inconsistent monosaccharide composition analyses being detailed thus far (Chai et al ., 2012 ; Jones et al ., 2014 ; Roux et al ., 2015 ). The second component is the protein BslA that is responsible for the non‐wetting nature of the biofilm (Kobayashi and Iwano 2012 ; Hobley et al ., 2013 ; Bromley et al ., 2015 ) and for biofilm architecture, independently of its ability to render the surface of the biofilm water‐repellent (Arnaouteli et al ., 2017 ). The third component of the biofilm matrix is the protein TasA (together with accessory protein TapA) that is needed for biofilm structure including attachment to plant roots (Branda et al ., 2004 ; Romero et al ., 2011 ; Beauregard et al ., 2013 ). TasA is a product of the tapA‐sipW‐tasA locus (Michna et al., 2016 ). It is post‐translationally modified by SipW (Stöver and Driks, 1999 ), a specialized signal peptidase that releases the mature 261‐amino acid TasA into the extracellular environment where it forms long protein fibres that contribute to the superstructure of the biofilm matrix and are needed for biofilm integrity (Branda et al ., 2006 ; Romero et al ., 2010 ). In addition to functions involved in the process of biofilm formation, TasA is also linked with sliding motility (van Gestel et al ., 2015 ) and spore coat formation (Stöver and Driks, 1999 ; Serrano et al ., 1999 ). TasA fibres can be extracted from B. subtilis biofilms, and exogenous provision to a tasA null strain has previously been reported to reinstate structure to floating pellicles (Romero et al ., 2010 ). Due to the reported ability of TasA fibres to bind the dyes Congo Red and Thioflavin T (ThT), ex vivo purified TasA fibres have previously been classified as functional bacterial amyloid fibres (Romero et al ., 2010 ), placing them alongside the curli fibres of E. coli (Chapman et al ., 2002 ). Amyloid‐like fibres are well‐known for their association with diseases like Alzheimer's and Parkinson's (Eisenberg and Jucker, 2012 ). In these conditions, highly stable fibrillar protein deposits are found in tissue sections, and are associated with cell damage (Hardy and Selkoe, 2002 ). The amyloid fibres in these deposits are characterised by several properties: (i) β‐sheet‐rich structures that are assembled into the canonical ‘cross‐β’ structure; (ii) the ability of the fibres to bind the dye Congo Red and exhibit green birefringence under cross polarised light; (iii) kinetics of formation that indicate a nucleated self‐assembly process and (iv) a fibril structure that is unbranched, 6–12nm in diameter, and often microns in length (Sunde and Blake, 1997 ; Sipe et al ., 2016 ). Once formed, these protein aggregates are highly stable, and in many cases are thought to be the lowest energy structural form shorter polypeptide chains can adopt (Baldwin et al ., 2011 ). ‘Functional’ amyloid fibres refer to structurally robust, protease and SDS resistant fibrillar protein deposits that share the characteristic structural properties of amyloid fibres, but are beneficial to the organism rather than being associated with disease (Fowler et al ., 2007 ). Significant caution is required in identifying functional amyloid‐like fibres from predominantly in vitro data however, as many proteins and peptides can be induced to adopt the canonical amyloid fibre cross‐β fold through appropriate manipulation of solution conditions such as changes in pH, temperature, cosolvent, salt or the presence of an interface (Kayed et al ., 1999 ; Ferrão‐Gonzales et al ., 2000 ; Uversky et al ., 2001 ; Hong et al ., 2006 ; Kalapothakis et al ., 2015 ), which may or may not be of physiological relevance. Indeed, the ability of proteins to assemble into the cross‐β architecture appears to be a ‘generic’ property of the polypeptide chain, independent of the amino acid sequence or the native structure of the precursor (Dobson, 1999 ; MacPhee and Dobson, 2000 ). Here, we show that, although TasA is a fibre‐forming protein, it is not amyloid‐like in character. We have produced recombinant TasA in both fibre and monomeric forms, and show that the secondary structures of these are both identical to each other and to those reported previously for the exogenous purified TasA fibres (Romero et al ., 2010 ; Chai et al ., 2013 ), appearing significantly helical in character. We have also examined native TasA fibres in enriched extracts from B. subtilis and show that both the native and recombinant forms of fibrous TasA show indistinguishable biological activity, being able to reinstate biofilm structure to a Δ tasA sinR deletion strain. X‐ray fibre diffraction of the recombinant TasA fibres shows that they are assembled from a helical repeat of globular protein units arranged approximately 45 Å apart, and the data are not consistent with the canonical ‘cross‐β’ diffraction pattern associated with amyloid‐like fibres. Neither monomeric nor fibrous forms of recombinant TasA bind the dyes Congo Red or ThT, and although TasA‐enriched extracts from B. subtilis biofilms show both Congo Red and ThT binding activity, this is at a similar level to that produced by protein extracts from cells lacking tasA . Thus, TasA does not fall into the class of ‘functional amyloid‐like fibres’; nonetheless it plays a critical role in biofilm structure.",
"discussion": "Discussion We have demonstrated that recombinant fibrous TasA can return rugosity to a B. subtilis Δ tasA sinR deletion strain and shares the biological functionality of native TasA purified from B. subtilis . Biophysical analysis indicates that these fibres are assembled as a helical arrangement of globular units that lack the characteristic ‘cross‐β’ architecture of canonical amyloid‐like fibres. The CD spectrum of the recombinant protein resembles that published previously for native TasA isolated directly from B. subtilis (Romero et al ., 2010 ; Chai et al ., 2013 ) and is suggestive of a predominantly helical secondary structure. Moreover, we have demonstrated that recombinant TasA can be rendered monomeric by the addition of a single amino acid to the N‐terminus, and that this monomeric protein shares the same secondary structure as the fibrous form. This strongly suggests that the fibres comprise a linear assembly of these monomeric units, with no large structural rearrangement, although domain‐swapping between monomers cannot be ruled out. Indeed, a repeating unit is visible along the length of the fibre axis, most clearly in the TEM images of recombinant fibres of the orthologous TasA protein from B. cereus where the protein subunits appear horizontally aligned across a fibre bundle, but also visible in all forms of TasA we have examined. Such a structure is not consistent with current structural models of amyloid‐like fibrils, which comprise a single continuous hydrogen‐bonded array along the long axis of the fibril. Taken together, our data indicate that TasA is unlikely to fall into the class of functional amyloid‐like fibres. We further found that our recombinant forms of TasA did not bind either Congo Red or ThT dyes that are commonly used to assess the formation of amyloid‐like fibres. Moreover, our protein extracts from B. subtilis showed dye binding activity irrespective of whether TasA was present or not. Caution should be taken when inferring the formation of amyloid‐like fibres from enhanced fluorescence in the presence of ThT, which also exhibits enhanced fluorescence in the presence of globular proteins such as bovine serum albumin (Freire et al ., 2014 ), human serum albumin (Sen et al ., 2009 ) and acetylcholinesterase (De Ferrari et al ., 2001 ); in the presence of amorphous aggregates of lysozyme and bovine serum albumin (Yang et al ., 2015 ), and amorphous aggregates formed by a thrombin‐derived C‐terminal peptide (Petrlova et al ., 2017 ); and in the presence of non‐amyloid wormlike aggregates of an artificial dimer of an Aβ peptide (Yamaguchi et al ., 2010 ). Conversely, ThT does not exhibit enhanced fluorescence in the presence of, for example, cross‐β fibrils formed by poly‐L‐lysine (Benditt, 1986 ; LeVine, 1999 ). Congo Red is similarly promiscuous (Howie and Brewer, 2009 ), although the observation of green birefringence under cross‐polarisers is one of the identifying characteristics of amyloid deposits in vivo . Thus, Congo Red binding and enhanced ThT fluorescence should be considered only suggestive, but not indicative, of amyloid‐like fibre formation. The widespread nature of functional amyloid fibres in bacterial biofilms has been hypothesized, and a well‐characterised example is the curli fibres of E. coli , Enterobacter cloacae , and Salmonella spp (Evans and Chapman, 2014 ). These show a CD spectrum, dye‐binding behaviour, enhanced stability and proteolytic insensitivity that are consistent with an amyloid‐like β‐sheet structure, but solid‐state NMR data suggests an architecture comprising stacked β‐helical subunits (Shewmaker et al ., 2009 ), a structural motif commonly employed by bacteria (Kajava and Steven, 2006 ). Many amyloid‐like fibres formed in vitro from proteins associated with disease show an in‐register parallel cross‐β arrangement (Margittai and Langen, 2008 ); recently however native Tau filaments extracted from the brain of an Alzheimer's Disease patient have been demonstrated to form an elaborate mixed β‐helix/cross‐β structure formed of in‐register, parallel β‐strands (Fitzpatrick et al ., 2017 ). Thus, both cross‐β and β‐helix architectures may be characteristic of amyloid fibres, and curli fibres may still be considered as ‘amyloid‐like’. Making the correct distinction between amyloid‐like and non‐amyloid fibrous proteins is more than a semantic argument: a number of papers have drawn a link between functional amyloid‐like fibres formed by bacteria and their relevance to human disease (Epstein and Chapman, 2008 ; Chai et al ., 2013 ; Evans and Chapman, 2014 ), for example, in the determination of the mechanistic details of self‐assembly, or in the possible discovery of new therapeutics. As the amyloid‐like fibre macrostructure is thought to be a ‘generic’ property deriving from the chemical structure of the polypeptide backbone that is common to all proteins and peptides – and thus to a large extent independent of primary sequence, although this will influence overall fibre morphology – small drug molecules that target the generic amyloid fold may have widespread applicability in a number of devastating human diseases. Thus it is important to make the distinction between non‐amyloid fibrous assemblies and amyloid‐like fibres appropriately. The fibrous nature of TasA likely imparts mechanical rigidity to the biofilm, thereby restoring the highly wrinkled architecture characteristic of the ΔtasA sinR deletion strain. As indicated above it is unclear why neither fTasA nor nTasA(+) can recover biofilm architecture to the single tasA deletion and furthermore, why expression of a sipW‐tasA construct is required for genetic complementation. Since SinR has pleiotropic roles in biofilm formation (Vlamakis et al ., 2013 ; Cairns et al ., 2014 ) it may be that overproduction of the biofilm polysaccharide compensates for the loss of native regulation that intricately controls native TasA production in space and time (Vlamakis et al ., 2008 ). Our results also indicate that when in a fibrous form, TasA does not require the TapA protein to fulfil its function, which contradicts previous reports suggesting that TapA is an accessory protein required for correct TasA assembly and localisation (Romero et al ., 2011 ). Therefore the role played by TapA in biofilm formation, while evidently essential (Chu et al ., 2006 ), is unclear. It may be that while TapA is not essential for TasA fibre formation in vitro , it functions as a chaperone in vivo to aid the transition of monomeric TasA into a fibrous state. This hypothesis is consistent with the overall reduction in the level of TasA and the corresponding reduction in the number of TasA fibres observed in the tapA mutant (Romero et al ., 2011 ). Moreover, it is consistent with the demonstration that monomeric TasA, but not fibrous TasA, is susceptible to degradation by the extracellular proteases. A non‐amyloid‐like structure for TasA is possibly beneficial in the context of the B. subtilis biofilm; amyloid‐like self‐assembled fibres are very stable, with curli fibres, for example, requiring treatment with concentrated acid solutions to drive disassembly (Chapman et al ., 2002 ). Curli fibres also appear to form a brittle matrix which, once fractured, does not recover (Serra et al ., 2013 ). In contrast, we have shown that the gelation properties of fibrous TasA solutions recover after shear (Supporting Information Fig. S5D–F), suggesting that in vivo the biofilm matrix could be remodelled in response to mechanical environmental perturbations. The TasA fibres may also be in equilibrium with the monomeric form of the protein, which would allow dynamic restructuring of the biofilm in response to environmental changes. As the fibrous form of the protein confers protection against degradation by extracellular proteases whereas the monomeric protein is degraded, an appropriate secretion of monomeric protein and/or proteases could provide dynamic control of biofilm elasticity and structure."
} | 4,112 |
37255364 | null | s2 | 6,131 | {
"abstract": "Inspired by spider silk's hierarchical diversity, we leveraged peptide motifs with the capability to tune structural arrangement for controlling the mechanical properties of a conventional polymer framework. The addition of nanofiller with hydrogen bonding sites was used as another pathway towards hierarchical tuning "
} | 79 |
28480150 | PMC5415339 | pmc | 6,132 | {
"abstract": "Biobased\nfuranics like 5-hydroxymethylfurfural (5-HMF) are interesting\nplatform chemicals for the synthesis of biofuel additives and polymer\nprecursors. 5-HMF is typically prepared from C6 ketoses like fructose,\npsicose, sorbose and tagatose. A known byproduct is 2-hydroxyacetylfuran\n(2-HAF), particularly when using sorbose and psicose as the reactants.\nWe here report an experimental and kinetic modeling study on the rate\nof decomposition of 2-HAF in a typical reaction medium for 5-HMF synthesis\n(water, Brönsted acid), with the incentive to gain insights\nin the stability of 2-HAF. A total of 12 experiments were performed\n(batch setup) in water with sulfuric acid as the catalyst (100–170\n°C, C H 2 SO 4 ranging\nbetween 0.033 and 1.37 M and an initial 2-HAF concentration between\n0.04 and 0.26 M). Analysis of the reaction mixtures showed a multitude\nof products, of which levulinic acid (LA) and formic acid (FA) were\nthe most prominent ( Y max,FA = 24 mol %, Y max,LA = 10 mol %) when using HCl. In contrast,\nboth LA and FA were formed in minor amounts when using H 2 SO 4 as the catalyst. The decomposition reaction of 2-HAF\nusing sulfuric acid was successfully modeled ( R 2 = 0.9957) using a first-order approach in 2-HAF and acid.\nThe activation energy was found to be 98.7 (±2.2) kJ mol –1 .",
"conclusion": "Conclusions 2-HAF\nis a known side product from the acid catalyzed dehydration\nof C6 sugars to 5-HMF in water using Brönsted acid catalysts.\nFor optimization of the 2-HAF yields from C6-sugars, information about\nthe stability of 2-HAF in the reaction medium at relevant conditions\nis required. In this paper, the kinetics of 2-HAF decomposition using\nsulfuric acid as the catalyst in water have been determined. A good\nagreement between model and experimental data ( R 2 = 0.9957) was obtained when using a first-order approach\nin both 2-HAF and H + . The activation energy was 98.7 ±\n2.2 kJ/mol. At 170 °C, the reaction rate using HCl is slightly higher than for H 2 SO 4 and combined with the differences in product portfolio suggests that the anion plays a major role. The reaction does not lead\nto the formation of a single reaction product; instead, a multitude\nof soluble non-identified products was observed (HPLC) and solids formation\nwas also inevitable. The only exceptions are LA and FA, which were\npresent in significant amounts when using HCl as the catalyst ( Y LA = 10 mol % and Y FA = 24 mol %). The findings described in this paper will be of relevance\nfor the development of an efficient route for 2-HAF from C6 sugars and\nallow selection of optimum conditions to reduce the rate of 2-HAF decomposition.",
"introduction": "Introduction Biobased furanics like 5-hydroxymethylfurfural\n(5-HMF) are interesting\nplatform chemicals for the synthesis of biofuel additives and polymer\nprecursors like 2,5-furandicarboxylic acid and derivatives. 1 5-HMF is typically prepared from C6-sugars, with\na high preference for d -fructose. We have recently performed\nextensive experimental studies on the use of other C6-ketoses (fructose,\npsicose, sorbose and tagatose) for 5-HMF formation 2 − 5 in water using sulfuric acid as\nthe catalyst and it was shown that particularly sorbose is also a\ngood source for 5-HMF synthesis ( Scheme 1 ). Scheme 1 Reaction Scheme for the Acid Catalysed Hydrolysis\nof Sorbose in Aqueous\nSolutions Besides the target\ncomponent 5-HMF, considerable amounts of 2-hydroxyacetylfuran\n(2-HAF) or 2-furoylcarbinol were formed, the exact amount being a\nfunction of the ketose used. When using d -sorbose, the amount\nof 2-HAF was up to 10 mol %. 3 2-HAF is\npotentially an interesting biobased furanic compound with a high derivatization\npotential and activities to increase the 2-HAF yields from ketoses are\nin progress. 2-HAF was already reported as the side product\nof sucrose dehydration\nin acidic conditions in the 1950s. 6 , 7 Later studies\nshowed that it is also formed during the dehydration of the monomeric\naldoses like glucose 6 , 8 − 10 and mannose 11 and ketoses like fructose. 11 , 12 A number of studies have been performed to elucidate the mechanism\nof 2-HAF formation from C6 sugars. 8 , 9 , 12 − 15 It is postulated that 2-HAF is formed from d -fructose by an acyclic 2,3-enolization, which though is less favorable\nthan the direct dehydration after an 1,2 enolization to form 5-HMF\n( Scheme 2 ). Scheme 2 Proposed,\nSimplified Mechanism for the Acid Catalyzed Reaction of d -Fructose to 5-HMF and 2-HAF 8 , 9 , 12 , 14 , 15 To optimize the synthesis of\n2-HAF from C6 sugars, it is essential\nto gain insights in the stability of 2-HAF in the reaction medium\nand to obtain information about the reaction products, both qualitatively\nand quantitatively. We here describe an experimental study on the\nconversion of 2-HAF in water using sulfuric acid as the catalyst at\nconditions of relevance (100–170 °C, C H 2 SO 4 ranging between 0.033 and\n1.37 M, C HAF,0 between 0.04 and 0.26 M). The reaction mixtures\nwere analyzed with HPLC and GC/MS-FID for product identification.\nA kinetic model was developed and the kinetic parameters were determined.\nTo investigate possible Brönsted catalyst effects, a number\nof experiments with HCl were performed as well. With this information,\nthe rate of decomposition of 2-HAF can be determined as a function of\nprocess conditions and provide input in the research aimed to optimize\n2-HAF yields from various sugars.",
"discussion": "Results\nand Discussion 2-HAF Reactivity in Water Using H 2 SO 4 as\nthe Catalyst Screening Studies In the first stage\nof this study,\nthe effect of process conditions on the conversion of 2-HAF in water\nusing sulfuric acid as the catalyst was investigated in a batch setup.\nA total of 12 experiments was performed in a temperature window of\n100–170 °C, C H 2 SO 4 ranging between 0.033 and 1.37 M, and an initial 2-HAF\nconcentration ( C HAF,0 ) between 0.04 and\n0.26 M. A typical concentration–time profile for an experiment\nis shown in Figure 3 . Figure 3 Typical reaction profile for the acid-catalyzed decomposition of\n2-HAF at T = 170 °C, C H 2 SO 4 = 1.37 M, C HAF,0 = 0.14 M. After reaction, the solution was slightly yellowish, and\nin case\nof the experiments at more severe conditions, also contained some\nbrown solids (humins). The main\ndetectable soluble component was LA, though the amount was always\nless than 4 mol %. HPLC revealed the presence of numerous other peaks\nwith small intensities, of which none could be assigned unequivocally\n(see Supporting Information , Figure S1) When analyzing the reaction mixture with GC–MS, a peak at\na retention time of about 11 min was assigned by the GC–MS\nlibrary as butyrolactone (73% probability). However, spiking\nof a representative HPLC sample with butyrolactone, showed that the\nlatter was detected at a retention time of 26.1 min. The initial HPLC\nsample did not show this peak, a clear indication that butyrolactone\nis not formed during reaction. In conclusion, the results indicate\nthat 2-HAF is not stable under\nthe conditions employed during its synthesis from C6-ketoses. As such,\n2-HAF is an intermediate product and optimum reaction conditions need\nto be employed to maximize its yield. In this respect, there are strong\nresemblances with the synthesis of furfural from C5-sugars in water\nusing Brönsted acids as the catalyst. Here furfural is also\nprone to decompose to complex mixture of products and selection\nof proper reaction conditions to reduce the rate of furfural decomposition\nis of prime importance to obtain high furfural yields. In addition,\nit is clear that 2-HAF is not easily converted to LA and as such,\nis not a major source of LA when converting C6 sugars like for instance\nsorbose to 5-HMF. The effect of temperature, sulfuric acid concentration\nand initial\n2-HAF concentration on the decomposition rate of 2-HAF were determined,\nand the results are given in Figures 4 , 5 and 6 . It is evident that higher temperatures and sulfuric acid concentrations\nresult in higher decomposition rates of 2-HAF. In contrast, the conversion\nof 2-HAF is almost independent of the initial 2-HAF concentration\n( Figure 6 ), an indication\nthat the reaction order in 2-HAF is close to 1 ( vide infra ). Figure 4 Concentration of 2-HAF versus time at different temperatures ( C HAF,0 = 0.14 M, C H 2 SO 4 = 1.37 M). Figure 5 Concentration of 2-HAF versus time at different sulfuric acid concentration\n( C HAF,0 = 0.04 M, T =\n120 °C). Figure 6 Concentration of 2-HAF\nversus time at different initial 2-HAF concentration\n( C H 2 SO 4 = 1.37 M, T = 170 °C). LA was formed in detectable amounts only for the experiments\nperformed\nat relatively severe conditions, i.e. the highest sulfuric acid concentration\n(1.37 M) and temperatures of 140 °C and above. However, the yields\nof LA were always below 4 mol %, a clear confirmation that 2-HAF is\nnot a major precursor for LA formation. Development of a Kinetic\nModel The conversion of 2-HAF\nwas modeled based on the simplified reaction scheme given in Scheme 3 . Scheme 3 Simplified Reaction\nScheme for the Acid Catalyzed Decomposition of\n2-HAF The reaction rate was initially\nmodeled using a power-law approach;\nsee eq 6 for details. 6 The temperature\ndependency of the kinetic constant is defined in\nterms of a modified Arrhenius equation: 7 In this equation, T is the reaction temperature\nand T R is the reference temperature, which\nwas set at 140 °C for this study. The acid concentration is included\nin the reaction rates and calculated as follows 8 where K a,HSO 4 – is the dissociation constant of HSO 4 – , which\nwas calculated using eq 9 . 9 Here the p K a is calculated with eq 10 using a correction for\nthe temperature of\nthe mixture ( T ): 10 For a batch reactor setup,\nthe concentration\nof the 2-HAF as a function of time is represented by the following differential\nequation: 11 Modeling\nResults A total of 12 experiments gave 122\nexperimental data points that consist of the concentrations of 2-HAF\nat different batch times. The best estimation of the kinetic parameters\nand their standard deviations were determined using a MATLAB optimization\nroutine. The results when using the power-law model are given in the Supporting Information (Table S1). However, the\nvalues of the powers in the reactants (2-HAF and H + ) were\nclose to 1 for the power-law model and as such the number of model\nparameters was reduced by taking orders of 1 for both 2-HAF and H + ( a H = α HAF =\n1) in the model. Good agreement between model and experimental\nvalues was observed. This is evident from the R 2 of 0.9957 ( Table 1 ), the experimental and model graphs ( Figure 7 ) and a parity plot in Figure 8 . Table 1 Kinetic Parameter\nEstimation for Decomposition\nof 2-HAF using H 2 SO 4 as the Acid Catalyst Parameter Value R 2 0.9957 E 1X (kJ mol –1 ) 98.7 ± 2.2 k 1RX (M –1 min –1 ) a 0.032 ± 0.001 a The values were determined at a\nreference temperature ( T R ) of 140 °C Figure 7 Comparison of experimental data (○)\nand kinetic model (solid\nlines) for different initial 2-HAF concentrations, temperature and acid\ncatalyst concentrations. Figure 8 Parity plot with the experimental and corresponding model values\n( C HAF , M). The activation energy for the reaction is 98.7 kJ/mol. A\ncomparison\nwith literature data is difficult as no studies have been reported\nfor the decomposition reaction of 2-HAF. However, it is informative\nto compare the activation energy with those reported for the reaction\nof 5-HMF to either LA and/or humins. An overview is given in Figure 9 and detailed information\nis shown in Table 2 . Figure 9 Activation energies for the conversion of 2-HAF (black bar) and\n5-HMF (white bars: using H 2 SO 4 ) and other homogeneous\nacid catalysts (gray bars). Table 2 Overview of the Activation Energies\nfor the Conversion of 2-HAF and 5-HMF Using Several Homogeneous Acid\nCatalysts in Water Feed Acid E a (kJ mol –1 ) # Name C feed Name Concentration T (°C) 5-HMF or 2-HAF to LA 5-HMF or 2-HAF to humins ref 1 Glucose 0.0057–0.333 M Buffer: butyric acid/H 3 PO 4 and NaOH pH 1–4 170–230 56 n.d. ( 18 ) 2 Wheat 16:1 w/w water:wheat H 2 SO 4 1–5 w/w-% 190–230 56 51 ( 19 ) 3 5-HMF 5%-w/v H 2 SO 4 1–5 w/w-% 170–210 57 n.d. ( 20 ) 4 5-HMF 0.1–1 M H 2 SO 4 0.005–1 140–180 92 119 ( 21 ) 5 5-HMF n.d. HCl, subcritical water 1.8 210–270 94 122 ( 22 ) 6 5-HMF 0.06–0.14 M H 2 SO 4 0.025–0.4 N 160–220 97 n.d. ( 23 ) 7 Glucose 56–112 mM CH 3 COOH 5–20 w/w-% 180–220 107 127 ( 24 ) 8 5-HMF 0.1–1 M H 2 SO 4 0.05–1 M 98–181 110 111 ( 2 ) 9 Cellulose 49.8–149 mM HCl 0.309–0.927 M 160–200 144 147 ( 25 ) 10 2-HAF 0.04–0.26 M H 2 SO 4 0.033–1.37 M 100–170 n.d. 99 This study The data reveal that\nthe activation energy for the decomposition\nof 2-HAF is in the range as reported for that of 5-HMF to humins and\nwithin the range for 5-HMF to LA. However, a good comparison is\ndifficult as the activation energies from 5-HMF cover a large range\ndue to the use of various catalysts. When only considering the reactions\nwith sulfuric acid (white bars in the Figure 9 ), it can be concluded that the activation\nenergy for the decomposition of 2-HAF to humins is comparable with\nthat for 5-HMF to humins. For the optimization of the conversion\nof C6 sugars to either 2-HAF\nor 5-HMF, it is of interest to compare the relative stability of both\ncompounds under reaction conditions. In Figure 10 , the relative ratio of the reaction rates\nfor the decomposition of 2-HAF ( R 1,HAF ),\nas presented in this study, and those for 5-HMF ( R HMF,tot ) are provided. The data for 5-HMF were taken from an\nearlier publication of our group using sulfuric acid as the catalyst. 2 For 5-HMF, the reaction rate was the sum of the\nrate of reactions ( R HMF,tot ) to both\nLA ( R HMF,LA ) and humin ( R HMF,humin ). Figure 10 Ratio of reaction rates for 5-HMF and 2-HAF decomposition\nversus the\ntemperature ( C acid = 0.1 M, C HMF = C HAF = 0.25\nM). On the basis of these data, we\ncan conclude that 2-HAF is more stable\nunder the given reaction conditions than 5-HMF. Moreover, this effect\nis more pronounced at higher temperatures, in line with the lower\nexperimental activation energy found for the reaction of 2-HAF (99 kJ\nmol –1 ) compared to 5-HMF (110 kJ mol –1 ) when using sulfuric acid as the catalyst. 2-HAF Reactivity in Water\nUsing HCl as the Catalyst To gain insights in the role of\nthe Brönsted acid catalyst,\na number of exploratory experiments were carried out with HCl instead\nof sulfuric acid ( C HAF,0 = 0.14 M, C HCl = 1.37 M, T =\n170 °C). The concentration time profiles for 2-HAF and LA for both\ninorganic acids are provided in Figure 11 . Figure 11 Comparison of the concentration–time\nprofiles for the acid-catalyzed\ndecomposition of 2-HAF in water at 170 °C, C HAF,0 = 0.14 M using C H 2 SO 4 (left) and C HCl (right)\nat a concentration of 1.37 M. The conversion rate of 2-HAF was slightly higher when using\nHCl.\nThe kinetic constant at 170 °C for HCl was calculated from the\nconcentration time profile in Figure 11 using a first order approach in 2-HAF and H + and found to be 0.23 M –1 min –1 , which is slightly higher\nthan for sulfuric acid (0.16 M –1 min –1 ) at similar conditions.\nOf interest is the significantly higher concentration of LA and FA\nin the product mixture when using HCl as the catalyst. For this particular\nexperiment, the yield of LA was 10 mol %, and the FA yield was up\nto 24 mol %, the remainder being unidentified soluble products and\ninsoluble resinous compounds known as humins. On the basis of\nthe product composition, a tentative reaction network\nis proposed; see Figure 12 for details. It involves the formation of humins by acid-catalyzed\n(aldol) condensation reactions of the starting materials and subsequent\nreactions with intermediates. LA and formic acid may be formed from\nan intermediate α-hydroxy-keto-aldehyde, obtained by the ring\nopening of 2-HAF followed by an acid catalyzed rearrangement. However,\ndetailed mechanistic studies, beyond the scope of this paper, will\nbe required to strengthen this proposal. Figure 12 Proposed reaction network\nfor 2-HAF decomposition in aqueous media\nusing Brönsted acids. The differences in reaction rate and product composition\nbetween\nHCl and sulfuric acid indicate that the outcome of the reaction is\ndepending on the inorganic acid used as the catalyst for the reaction.\nBased on the fact that both acids are strong and as such the H + concentrations are about equal, the anion must play an important\nrole. Such anion effects also have been reported for Brönsted\nacid catalyzed furfural decomposition reactions in water. The activation\nenergy for HCl ( E a = 48.1 kJ/mol 26 ) was reported to be about half of that when\nusing H 2 SO 4 ( E a =\n83.6 kJ/mol 27 ). The authors explained these\nresults by assuming a difference in reaction mechanism for both acids\ndue to anion effects, involving a ring opening mechanism when using\nCl – versus a direct dehydration mechanism when using\nsulfuric acid. 28 − 31 Anion effects have also been reported for the conversion of 5-HMF,\nanother example of a biobased furanic, to LA and formic acid. For\ninstance, Yoshida et al. 32 reported on\nthe acid-catalyzed production of 5-HMF from d -fructose and\nthe subsequent rehydration to LA in subcritical water using both sulfuric\nacid and HCl as the catalysts. Remarkable differences in the rate\nof reaction were observed between both acids at similar pH values,\nwith HCl giving higher 5-HMF yields. In addition, the addition of salts\nlike NaCl and Na 2 SO 4 showed that Cl – ions accelerate the conversion of fructose to 5-HMF and the subsequent\nreaction of 5-HMF to LA whereas sulfate ions have an inhibiting effect\non the rehydration reaction to LA. However, to the best of our knowledge,\ndetailed mechanistic studies to explain and rationalize these anion\neffects on the stability of biobased furanics like furfural and 5-HMF\nhave not been reported to date."
} | 4,488 |
26632996 | PMC4668581 | pmc | 6,133 | {
"abstract": "Ecological networks such as plant-pollinator and host-parasite networks have structured interactions that define who interacts with whom. The structure of interactions also shapes ecological and evolutionary dynamics. Yet, there is significant ongoing debate as to whether certain structures, e.g., nestedness, contribute positively, negatively or not at all to biodiversity. We contend that examining variation in life history traits is key to disentangling the potential relationship between network structure and biodiversity. Here, we do so by analyzing a dynamic model of virus-bacteria interactions across a spectrum of network structures. Consistent with prior studies, we find plausible parameter domains exhibiting strong, positive relationships between nestedness and biodiversity. Yet, the same model can exhibit negative relationships between nestedness and biodiversity when examined in a distinct, plausible region of parameter space. We discuss steps towards identifying when network structure could, on its own, drive the resilience, sustainability, and even conservation of ecological communities.",
"discussion": "Discussion We analyzed a nonlinear model of phage-bacteria dynamics ( Eqs. (1)-(2) , ) as a means to investigate the entanglement between interaction network structure, life history traits, and biodiversity. Given the complexity in the space of possible networks, we considered ensembles of networks that varied in a particular structural feature – nestedness – such that interaction ranges differ in the extent to which they form partially ordered subsets of one another. We found that there is not one globally applicable, positive relationship between biodiversity and nestedness in this model ( Fig. 3 ). Instead, we identified distinct regions in parameter space where there are contrasting relationships, both positive and negative ( Figs 4 and 5 ). Elevated nestedness is a common feature of interaction networks spanning both plant-pollinator and phage-bacteria systems 12 13 . Moreover, prior theoretical work has suggested that ecological communities whose interaction networks are nested are more likely to have higher relative biodiversity 12 . Our results highlight the need to understand the life history context underlying a given biodiversity-nestedness relationship. The totality of parameter space includes a subspace of biologically plausible values. Such subspaces often have relatively uninformative prior distributions. Therefore, using biologically plausible regions to restrict the parameters of a model is not a strong enough restriction to uniquely define the effect of network structure on community dynamics. Recently, it has been pointed out that different model parameterizations can lead to different biodiversity levels and consequently to contradictory results 20 . We expand on this point to show that this is also the case for whole regions of parameter space. This is important because studies using numerical simulations often average over different parameterizations. Indeed, Rohr et al. 20 examine to what extent parameters can vary for a given network structure before the community make-up changes. In contrast, our work examines how changes in network structure affects community make-up for a fixed set (or region) of parameters. These approaches are related, but they are not the same. Our results show how averaging over parameterizations is not sufficient to account for the effects of life history traits and that a more systematic study of parameter space is necessary. Additionally, we make a stronger case for the effect of parametrization by showing that it is possible to not only maximize biodiversity for specific networks but to obtain completely different trends of biodiversity vs. nestedness ( Fig. 6 ). In our view, statistical inference from numerical simulations can be informative and even advantageous, so long as certain precautions are kept in mind. The key point is that systematic analysis of parameter space is necessary whenever a numerical approach is used to characterize network structure-biodiversity relationships in nonlinear ecological systems. Studies that rely on analytical methods to estimate biodiversity or related features usually focus on fixed-point equilibrium states that are stable either locally or globally. This could be problematic for two reasons. First, general analytical solutions could be hard to find or interpret. Second, coexistence in high-dimensional ecological models could be characterized by non-equilibrium steady states. In such circumstances, fixed-point analyses would overlook configurations that are ecologically relevant. In the case of phage-bacteria dynamics, our study highlights the value of additional measurements of life history traits, complementary to the recent focus on methods for characterizing who infects whom 21 . We used a particular phage-bacteria model to highlight the importance of systematically studying model parametrization in distinct regions to better understand the relationship between network structure and biodiversity - yet the findings are relevant to a wider debate. The current findings point to the need to revisit the relationship between network structure, life history traits and biodiversity in other systems and given other kinds of network patterns. Optimistically, the systematic study of model parametrization could be of service in resolving ongoing debates concerning the relationship, or relationships, between biodiversity and network structure in plant-pollinator systems 14 15 20 22 ."
} | 1,390 |
34870016 | PMC8638023 | pmc | 6,136 | {
"abstract": "Biogenic coalbed\nmethane (CBM) is generally believed to be formed\nby anaerobic bacteria and methanogens, while a few studies took fungi\ninto account. Here, the microflora consisting of fungi and methanogens\nwas enriched from the produced water associated with the Qinshui Basin\nusing anthracite as the only carbon source. The maximum methane yield\nof 231 μmol/g coal was obtained after 22 days of cultivation\nunder the optimum temperature of 35 °C, pH of 8, salinity of\n0–2%, particle size of 0.075–0.150 mm, and the solid–liquid\nratio of 1:30. It could remain active even after exposure to air for\n24 h. Miseq results showed that the archaea were mainly composed of Methanocella , a hydrogenotrophic methanogen, followed by\nacetoclastic methanogen Methanosaeta and Methanosarcina , which could use various methanogenic substrates.\nThe fungal communities mainly included Amorphotheca , Alternaria , Aspergillus , and Penicilium , which are all able to degrade complex organics\nsuch as aromatics and lignin. After cultivation, the crystal structure\nof anthracite became looser, as shown by XRD results, which might\nbe due to the swelling effect caused by the destruction of the aromatic\nring structure of coal under the function of fungi. The stretching\nvibration intensity of each functional group in coal decreased with\ncultivation, as revealed by FTIR. The GC-MS results showed that the\nconcentration of alkanes and alcohols decreased significantly, which\nare the products of ring-opening of aromatics by fungi. These results\nsuggested that fungi and methanogens in the coalbed also can syntrophically\ndegrade coal effectively, especially for aromatics in coal.",
"conclusion": "3 Conclusions The\nmicroflora with fungi and methanogens was enriched from produced\nwater from the Qinshui Basin in this study. The microflora could degrade\nanthracite to generate methane with the maximum methane yield of 231\nμmol/g coal after 22 days of cultivation under the optimum conditions;\nthe temperature, pH, salinity, particle size, and the ratio of solid–liquid\nwere 35 °C, 6–9, 0–2%, 0.075–0.150 mm (100–200\nmesh), and 1:30, respectively. It can maintain methane generation\nactivity even after exposure to air for 24 h. According to the results\nof Miseq, the archaea were mainly composed of Methanocella , followed by Methanosaeta and Methanosarcina , showing that the methanogenic pathways were mainly hydrogenotrophic\nmethanogenesis. Amorphotheca , Alternaria , Aspergillus , and Penicilium were\ndominant fungi, which were able to degrade aromatic and lignin-derived\ncompounds in coal. XRD analysis showed that the aromatic compounds\nwere degraded effectively by fungi in the microflora, which would\ncause a swelling effect, making the crystal structure of anthracite\nlooser. After biodegradation, the stretching vibration intensity of\neach functional group in coal decreased; GC-MS results showed that\nthe concentration of alkanes and alcohols in the culture decreased\nsignificantly during methane production, which are the products of\naromatic biodegradation by fungi. These results suggested that the\nmicroflora with fungi and methanogens enriched from the produced water\ncould degrade anthracite and generate methane, especially ferment\naromatic compounds in coal effectively. They also provided a new way\nto better understand the mechanism of biogenic CBM formation and bioconversion\nof high-rank coal.",
"introduction": "1 Introduction In\nrecent years, the exploitation and development of unconventional\nnatural gas has been widely carried out to meet the increasing energy\ndemand. 1 As an important unconventional\nnatural gas, CBM refers to the methane adsorbed in coal seam, 2 which is being vigorously developed in the major\ncoal-producing countries of the world. Biogenic CBM is an important\npart, which accounts for more than 20% of CBM worldwide and an additional\n10% may also be of microbial origin. 3 , 4 Most of the\nbiogenic CBM retained in coalbed nowadays is secondary, which is generated\nafter coal formation by the anaerobic microorganisms in coalbed under\nsuitable environmental conditions. 5 , 6 Based on the\nformation of biogenic CBM, the technology of microbially enhanced\nCBM (MECBM) was proposed to convert coal into CH 4 via specific\nmicroorganisms. It can not only biomine raw coal and residual coal\nbut also increase the porosity of coal and decrease the affinity of\ncoal for CH 4 to improve the development of CBM. 7 In addition, other organic liquid products could\nalso be generated from coal during MECBM, which further added to its\nvalue. 8 , 9 As coal is a complex organic compound,\nwhich is composed of aromatic\nhydrocarbons, aliphatic hydrocarbons, and heterocyclic compounds,\nvarious microorganisms with diverse metabolic characteristics are\nrequired to degrade coal to produce methane. 10 Only one paper reported methane production from coal by a single\nmethanogen. 11 It is generally believed\nthat microorganisms mainly break the coal structure and metabolize\nthe intermediates such as fatty acids, organic acids, and alcohols\nto generate substrates for methanogens. 12 Most of the previous studies analyzed the methanogens and bacteria\nthat functioned in coal biodegradation. Various methanogens have been\ndetected in the produced water and the culture solution in the laboratory,\nsuch as acetoclastic Methanoseta , hydrogenotrophic Methanobacterium , Methanocella , and methylotrophic Methanolubus . Proteobacteria , Firmicutes , Clostridiales , Actinobacteria , and Bacteroidetes are found to be the dominant\nbacteria in CBM fields and the main participant in coal degradation. 3 , 13 − 17 Several microflora consisting of bacteria and methanogens have also\nbeen enriched from the produced water. 18 − 20 Jones et al. enriched\na methanogenic mixed culture from wetland named WBC-2 with the ability\nto degrade coal, which was mainly composed of Clostridium sp., Bacteroides spp., Acetobacterium sp., and acetoclastic methanogen Methanomicrobia . 21 A great number of evidence have\nshown that fungi are good degrader\nfor macromolecular compounds such as lignin, lignocellulose, lignin-derived\ncompounds, synthetic dyes, and polycyclic aromatics (PAHs). 22 , 23 Some fungi have also been employed to ferment coal. Fungi flora\nAD-1, isolated from the low rank coal, was found to have the abilities\nof decarboxylation and deamination, as well as breaking the side chains\nof the aromatic rings. 24 Trichoderma atroviride was used to biosolubilize\ncoal in a stirred tank reactor. 25 Polyporus versicolor and Poria monticola , two species of basidiomycete fungi, were reported to attach to\nthe coal surface and liquefy and biodegrade lignite. 26 Haider et al. isolated fungi MW1 to pretreat the low rank\ncoal to improve the production of humic acids. 27 Actually, the syntrophic relationship between anaerobic\nfungi and methanogens has been detected in methane production in bovine\nrumen, and the microflora consisting of anaerobic fungi and methanogens\nhas also been isolated with the high fiber degradation ability. 28 , 29 However, only a few studies reported the fungi communities related\nto the formation of biogenic CBM. 30 , 31 A surprising\ndiversity of the fungal community was found in the produced water\nfrom the Qinshui Basin. The main fungi included Rhodotorula , Mortierella , Acremonium , Fusarium , Trichoderma , Aspergillus , and Schizophyllum , which could generate a significant\nmethane yield from coal in collaboration with methanogens. 30 In the laboratory, fungi were also found to\nplay a role in coal degradation together with bacteria and methanogens. 31 Thus, the fungi community are the non-negligible\npart in coal biodegradation to produce methane. In this study,\na microflora with fungi and methanogens to degrade\nanthracite and produce methane was enriched from the produced water\nobtained from the Qinshui Basin. The fungal and archaeal communities\nin the microflora were evaluated using Miseq. The growth conditions\nof microflora were optimized. The success of the coal structure and\nintermediates during methane production was analyzed by Fourier transform\ninfrared spectrometry (FTIR), X-ray diffraction (XRD), and gas chromatography-mass\nspectrometry (GC-MS) to discuss the mechanism of coal biodegradation\nby the microflora.",
"discussion": "2 Results and Discussion 2.1 Physicochemical Properties of Coal and Formation\nWater The proximate analysis and ultimate analysis of coal\nsamples showed that M ad , A ad , and V daf were 1.90, 10.41, and 8.82%, respectively, and C d , H d , O d , N d , and S td were 83.01,\n3.24, 1.76, 1.27, and 0.31%, respectively, which belonged to the range\nof anthracite. 32 The concentrations of\nmajor cations, anions, and ammonium in the formation water are shown\nin Table 1 , which are\nsimilar with the previous research in the same site; both detected\nhigh salinity with high concentration of Na + . 33 Table 1 Analysis Result of\nFormation Water\nSamples parameter units values K + mmol/L 0.02 Na + mmol/L 18.01 Ca 2+ mmol/L 0.12 Mg 2+ mmol/L 0.01 Cl – mmol/L 1.36 SO 4 2– mmol/L 0.32 NO 3 – ×10 –3 mmol/L 0.48 NO 2 – ×10 –3 mmol/L 0.22 TOC mmol/L 0.14 2.2 High Methane Yielded by\nthe Microflora with\nFungi and Methanogens The methanogenic flora with fungi and\nmethanogen was successfully enriched from the produced water with\nthe ability to degrade coal and produce methane. Figure 1 shows the methane productions\nin the 1st, 5th, 10th, 15th, and 20th generations of enrichment. With\ntransferring, the time for the microflora to reach the maximum methane\nyield decreased and the methane production became constant. The maximum\nmethane yield of about 220 μmol/g coal was reached on day 20\nincubation after enrichment, suggesting that fungi in produced water\ncould degrade the high-rank coal such as anthracite to supply substrates\nfor methanogens to produce methane. Figure 1 Methane productions in 1st, 5th, 10th,\n15th, and 20th generations. 2.3 Phylogenetic Composition of Microbial Communities\nin Microflora The summary of fungal and archaeal MiSeq reads,\nOTUs, and diversity estimators of enriched microflora at 97% similarity\nare shown in Table 2 . A total of 32 018 fungal sequence reads and 39 691\narchaeal sequence reads were generated by MiSeq, and 33 fungal OTUs\nand 15 archaeal OTUs were obtained. The coverages were both above\n0.999. The Shannon and Simpson estimators showed that the fungal diversity\nwas more than two times higher than archaeal diversity. Table 2 Summary of Archaeal and Fungal MiSeq\nReads, OTUs, and Diversity Estimators of Enriched Microflora at 97%\nSimilarity reads OTUs Ace Chao1 Shannon Simpson coverage fungi 31 887 33 33.00\n(33.00, 33.00) 33.00 (33.00, 33.00) 1.84\n(1.83, 1.85) 0.25 (0.25, 0.25) 0.9999 archaea 39 169 15 16.00 (15.13, 22.46) 15.00 (15.00, 0.00) 0.76 (0.75, 0.77) 0.68 (0.67, 0.68) 0.9999 The phylogenic composition of communities\nis shown in Figure 2 . There are five\nmain fungal genera with more than 5% of sequence reads including Amorphotheca (42.95%), Alternaria (21.62%), Aspergillus (9.13%), Penicilium (7.25%),\nand Malassezia (5.7%). They all had potential to\ndegrade aromatic and lignin-derived compounds in coal. 34 Amorphotheca is known to utilize\ndifferent kinds of organic substances such as alkanes, acetic acid,\nand lignocellulose biomass. 35 It has been\nconfirmed that Amorphotheca can degrade various inhibitor\ncompounds such as a high level of acetic acid from pretreated lignocellulose\nfeedstock, and it has been applied for producing ethanol, lactic acid,\ngluconic acid, and microbial lipid with a high product yield and zero\nwastewater generation. 36 − 38 Alternaria is one of only a few\nof fungi reported so far to be capable of degrading heavy crude oil,\nwhich is composed of PAHs and has a higher metabolization of the aromatic\nfraction. 39 Aspergillus was reported to use polysaccharide wastes as substrates to produce\nVFAs, acetate, and butyrate, and promote H 2 generation. 40 , 41 The growth of Aspergillus was also found on the\nsurface of aromatic polyesters, 42 demonstrating\nits remarkable ability to degrade complex aromatics, especially the\nhigh-molecular-weight PAHs. 43 Previous\nstudies have also shown that Aspergillus can degrade\ncarbohydrates in plants and rice straw. 44 Penicillium is a typical fermenter that can degrade\ncellulose and lignin effectively, 45 and\nit was also be found to efficiently degrade PAHs such as phenanthrene, 46 fluorene, 47 and pyrene. 48 Figure 2 Phylogenetic compositions of (a) fungal and (b) archaeal\ncommunities\nat the genetic level in the enriched microflora based on the MiSeq\ndata. The genera that contained <1% of the sequence reads were\ngrouped into “Others”. The archaea mainly included Methanocella (81.29%), Methanosaeta (12.54%), and Methanosarcina (3.55%). It suggested that the main metabolic type of the enriched\nconsortia was hydrogenotrophic methanogenesis, as Methanocella is a thermophilic hydrogenotrophic methanogen. 49 Methane could also be generated by other methanogenic pathways\nconsidering that the metabolic type of Methanosaeta is acetoclastic, and Methanosarcina can utilize\ndifferent substrates, such as CO 2 , H 2 , acetate,\nand methylamine. 20 , 31 Thus, the enriched microflora\ncan produce methane through diverse metabolic pathways, while hydrogenotrophic\nmethanogenesis is the main pathway. 2.4 Effects\nof Culture Conditions on Methane Production The most critical\nstep in the technology of MECBM was to obtain\nthe high-efficiency methanogenic microflora. The culture conditions\nwere one of the main factors influencing the efficiency of coal biodegradation\nby microflora. Thus, five critical factors were evaluated here, including\ntemperature, salinity, particle size, pH, and the solid–liquid\nratio. At the same time, the effect of the oxygen content was also\nevaluated. Methane production was significantly affected by\nthe culture temperature. As shown in Figure 3 a, the highest methane yield was 231 μmol/g\ncoal at 35 °C, while methane yields were 199 μmol/g coal\nat 25 °C and 58.47 μmol/g coal at 45 °C. However,\nat the initial stage, especially the first 5 days, the methane generating\nrate at 25 and 35 °C (both about 12 μmol/g coal per day)\nwas lower than that at 45 °C (48 μmol/g coal per day).\nIt showed that the higher temperature could enhance the production\nof methane in the early time. 50 Simultaneously,\nthe higher temperature also shortened the reaction period, which was\nabout 33 days at 25 °C, 19 days at 35 °C, and 12 days at\n45 °C. Figure 3 Methane produced by the microflora under different culture conditions,\nincluding (a) temperature, (b) pH, (c) salinity, (d) coal particle\nsize, (e) solid–liquid ratio, and (f) oxygen content. The cultivations can be divided into two groups\naccording to methane\nyields when pH changed from 4 to 10 ( Figure 3 b). When pH = 6–9, methane yields\ndid not change significantly, and 209.08–226.95 μmol/g\ncoal methane was observed when the maximum methane yield was obtained\nat pH = 8. When pH = 4, 5, and 10, methane yields were only 160, 177,\nand 160 μmol/g coal, respectively. The activity of microorganisms\nwas inhibited under higher acidity or alkalinity. 51 The microflora was more suitable for the weak alkalinity\ncondition. The salinity tolerance of the microflora was as high\nas 4% ( Figure 3 c).\nThe optimum salinity\nrange for coal degradation was 0–3%, and the highest methane\nyield of 230 μmol/g coal was observed at a salinity of 0.5%.\nWith the increase in salinity, the maximum methane production decreased,\nwhich was consistent with the previous study. 52 The methane production decreased significantly at a 4% salinity\nwith only 50 μmol/g coal, and even no methane was detected at\n5–6% salinity. The good salinity tolerance of the microflora\nis related to its origin habitat where a high concentration of Na + was detected ( Table 2 ). There was also an optimal coal particle size for\nmethane production.\nA maximum methane yield of about 231 μmol/g coal was obtained\nwhen the coal particle size was in the range of 0.075–0.150\nmm (100–200 mesh) ( Figure 3 d). When the coal particle size was >0.425 and <0.075\nmm, methane yields were both lower with only 159 μmol/g coal.\nIt did not agree with the previous study, which showed that the smaller\nthe coal particle size, the higher the methane production. 50 It was believed that the surface area of coal\nincreased with the decrease in coal particle size, leading to the\nincrease of contact between organisms and coal. However, too small\ncoal particles (<0.075 mm) would result in the reintegration of\ncoal particles, leading to a decrease in the contact area and low\nmethane production. Methane production was negatively correlated\nwith the solid–liquid\nratio ( Figure 3 e).\nThe maximum methane production of 231 μmol/g coal was observed\nat a solid–liquid ratio of 1:30. When the solid–liquid\nratio was 1:5 (6 g coal), the methane production was only 38 μmol/g\ncoal. The higher coal loading capacity would bring more toxic substances\ninto the culture, which inhibited the microbial activities, leading\nto a decrease in methane production. 53 It is hard to keep the microflora away from air all the time when\ninjecting it into coal seam. Thus, oxygen resistance is essential\nfor the application of microflora. As shown in Figure 3 f, methane production decreased with the\nprolongation of exposure time in the air. In the anaerobic environment,\nthe microflora could show the best biodegradation ability, and the\nhighest methane yield was 237 μmol/g coal. When the exposure\ntime was 3 h, the methane yield was about 200 μmol/g coal. After\nexposure for 6 h, it decreased to 170 μmol/g coal and continued\nto decrease to 150 μmol/g coal after 12 h and 115 μmol/g\ncoal after 24 h. It is expected that coal biodegradation could be\nperformed aerobically as many fungi detected here are facultatively\nanaerobic. At present, most methanogens are generally believed to\nbe strictly anaerobic, 54 , 55 while some methanogens are reported\nto survive in the oxygen environment for several hours and days with\nvery low methane generating rates. 56 − 58 Here, the microflora\nwere active even after 24 h of exposure to air, although the methane\nproduction was half of the maximum. The methane generating trend did\nnot change under different oxygen exposure times. The only difference\nwas the amount of methane produced, suggesting that the microbial\nactivities decreased after oxygen exposure. Thus, it can be believed\nthat methane production would be recovered after several transfers\nand the microflora is adaptable in the field. These results supported\nthe feasibility of injecting microflora into the coal seam. 2.5 Dynamic Changes in the Coal Crystal Structure\nduring Anaerobic Biodegradation The XRD spectra of raw coal\nand residual coal after 15 and 27 days of cultivation are shown in Figure 4 , and the corresponding\nstructural parameters are shown in Table 3 . In general, the change in the coal structure\nmainly occurred before 15 days, and significant changes were not detected\nbetween 15 and 27 days, which was consistent with methane production,\nwhich was almost completed at 15 days. The stacking height ( L c ), aromatic layers ( N ), and L a / L c increased with\nculture time. It can be inferred that the interaction of coal and\nmicroflora caused the swelling effect on the coal crystallite structure. A 26 and A 20 are believed\nto be equal to the number of aromatic carbon (C ar ) atoms\nand aliphatic carbon (C al ) atoms. 59 The intensity ratio ( A 26 / A 20 ) of the two peaks can reflect the aromaticity of coal. 39 During the reaction, A 26 and A 20 decreased from 11 706\nto 8547 and 1269 to 1185, respectively, suggesting that the number\nof aromatic and aliphatic carbon atoms decreased by 26.98 and 6.6%.\nThe ratio of A 26 / A 20 decreased from 9.22 to 7.21, indicating that the aromaticity\nand the ordered degree of the crystallite structure decreased. It\ncan be inferred that the fungal flora acted on aromatic parts of coal,\nbreaking the connection between aromatic rings and degrading the aromatic\ncompounds effectively. Figure 4 XRD curves of raw coal (RaC) and residual coal after 15\ndays (ReC15d)\nand 27 days (ReC27d) of cultivation. Table 3 Microcrystalline Structure Parameters\nof Coal during Degradation sample d 002 /nm L c /nm L a /nm N L a / L c A 26 A 20 A 26 / A 20 RaC 0.35 1.63 10.8 4.65 6.62 11 706 1269 9.22 ReC15d 0.34 2.02 9.75 5.94 4.83 11 575 1241 8.52 ReC27d 0.34 2.18 10.48 6.3 4.78 8547 1185 7.21 2.6 Changes of Functional Groups in Coal during\nAnaerobic Biodegradation The FTIR spectra of coal samples\nare shown in Figure 5 a. Similar to the results of XRD, the FTIR spectra of coal samples\nchanged significantly between 0 and 15 days, which showed that almost\nall of the bands decreased sharply, while they barely changed between\n15 and 27 days. The wavelengths between 3000 and 700 cm –1 were divided into three regions according to the previous studies. 60 , 61 The wavelengths between 900 and 700 cm –1 are attributed\nto aromatic groups, those between 1800 and 1000 cm –1 are attributed to oxygen-containing groups, and those between 3000\nand 2700 cm –1 are attributed to aliphatic hydrocarbon\ngroups. Their curve-fitting FTIR spectra are shown in Figure 5 b–d. The details for\neach adsorption band in all three groups, including the center position,\nheight, area, area %, and the assignment, are listed in Tables S1–S3 . Figure 5 FTIR spectra and curve-fitting\nFTIR spectra of coal samples including\nraw coal (RaC) and residual coal after 15 days (ReC15d) and 27 days\n(ReC27d) of cultivation. (a) FTIR spectra. (b) Aromatic functional\ngroups. (c) Oxygen-containing functional groups. (d) Aliphatic functional\ngroups. There were primarily six absorption\nbands detected in the infrared\nspectrum of aromatic structures ( Figure 5 b). The most apparent stretching vibration\nintensity of aromatic functional groups in raw coal (RaC) is the stretching\nvibration of aromatics with two substitutions, which accounted for\n27.43% of the aromatics. After 27 days of biodegradation, the stretching\nvibration intensity of each characteristic peak in the aromatic functional\ngroup weakened obviously when the range of peak intensity decreased\nfrom 4.17–11.94 to 1.3–6.11, which showed a correlation\nwith the decrease of A 26 in XRD that the\naromatic carbon decreased. It suggested that the fungi in microflora\ncould destroy the complex aromatic macromolecules in coal and utilize\nthe simple aromatic substances in coal to generate the substrate of\nmethane production, 62 , 63 leading to the depolymerization\nof coal and conducive to the follow-up biological reaction. Eight absorption bands were found in the region of oxygen-containing\nfunction groups ( Figure 5 c). The stretching vibration intensity of each functional group decreased\nobviously during biodegradation, especially for aliphatic with an\noxygen functional group, with the peak intensity of C–O alcohols\nand C–O ethers decreased by 81.17 and 86.38%. It is expected\nbecause oxygen-containing groups in coal are believed to be the acting\nsite for microorganisms. 64 These results\nsuggested that the oxygen-containing groups were also the favorite\nparts for fungi in microflora, which was similar to bacteria. 63 The curve-fitting infrared spectra of\naliphatic functional groups\nin the raw coal and degraded coal are shown in Figure 5 d, which were mainly divided into 10 adsorption\nbands. The most obvious changes in this region were the sym. R 2 CH 2 , which decreased from 22.75 to 6.51, and asym.\nRCH 3 , which increased from 12.28 to 18.17. The CH 2 /CH 3 (2920/2950 cm –1 ) ratios are often\nused to estimate the length and degree of branching of aliphatic side\nchains. 65 During the biodegradation, the\nstretching vibration of −CH 3 was weaker than −CH 2 , leading to the decrease in the ratio of CH 2 /CH 3 from 2.95 to 2.64, which suggested that the aliphatic chains\nin coal were degraded by the microorganism, resulting in the aliphatic\nchains becoming shorter or less branched. 2.7 Evolution\nof Intermediate Metabolites during\nMicrobial Degradation of Coal Figure S1 shows the GC-MS chromatogram of organics on 0, 15, and 27\ndays of cultivation. There were 20 prominent peaks divided into seven\ncategories: aliphatic acids, aromatic acids, heterocyclic compounds,\naliphatic alcohol, aliphatic ester, alkanes, and aromatic compounds\n( Figure 6 ). The area\nof each peak was analyzed by NIST. The percentage of different kinds\nof organic compounds in 20 peaks was calculated. The mechanism of\nfungi biodegradation of coal was further explored by analyzing the\npercentage changes of different organic components. The aromatics,\naliphatics, and alkanes were the main parts of the organic matter.\nIt was found that the alkanes in coal continuously degraded to produce\nmethane, decreasing from 35.45 to 8.11%. This change was consistent\nwith the decreasing trend of alkanes side rings [(CH) n , n > 4] in FTIR and bigger than\nthat caused by bacterial degradation, 63 indicating that fungi have a better ability to degrade alkanes than\nbacteria. The aromatic compounds in the culture only changed from\n15.96 to 18.71%. It seems that the release of aromatics from coal\nwas kept in balance with the transformation of aromatics in culture\nby the dominant fungi in the microflora, Alternaria , Penicillium , and Aspergillus ,\nwhich all have good ability to degrade aromatic compounds. In addition,\nthese fungi would also degrade aromatic compounds by the ring-opening\nway, 66 resulting in the generation of alkanes,\nheterocyclic compounds, aliphatic alcohols, and aliphatic ester. This\nmight be the reason for the reduction of aliphatic alcohols from 19.56\nto 1.6% during methane production. Figure 6 Classification of organic compounds and\ntheir distributions in\nculture on 0, 15, and 27 days of coal biodegradation. It is believed that methane production is restricted by the\ntoxic\nmatter released from coal at a later stage of coal biodegradation. 53 However, fungi would still act on the coal matrix\nin this process because they are more tolerant of harsh environments,\nleading to the accumulation of some intermediates during methane production.\nThe GC-MS results showed that aromatic acids, aliphatic acids, and\naliphatic esters were accumulated at the later stage from 15 to 27\ndays when methane production was completed. The proportion of aromatic\nacids, aliphatic acids, and aliphatic esters increased from 0, 3.39,\nand 14.4% in the initial stage to 8.74, 14.03, and 31.95% at the end\nof cultivation. These results suggested that aromatic acids, aliphatic\nacids, and aliphatic esters were more likely to be the key intermediates\nin coal biodegradation by fungi. This is consistent with the enriched\nfungal properties that Amorphotheca can degrade lignocellulose\nand other components to produce acidic substances and ester substances,\nand Aspergillus can degrade aromatic substances to\nproduce acid substances."
} | 6,780 |
33381099 | PMC7768010 | pmc | 6,137 | {
"abstract": "Volcanic areas emit a number of gases including methane and other short chain alkanes, that may serve as energy source for the prevailing microorganisms. The verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV was isolated from a volcanic mud pot, and is able to grow under thermoacidophilic conditions on different gaseous substrates. Its genome contains three operons encoding a particulate methane monooxygenase (pMMO), the enzyme that converts methane to methanol. The expression of two of these pmo operons is subjected to oxygen-dependent regulation, whereas the expression of the third copy ( pmoCAB3 ) has, so far, never been reported. In this study we investigated the ability of strain SolV to utilize short-chain alkanes and monitored the expression of the pmo operons under different conditions. In batch cultures and in carbon-limited continuous cultures, strain SolV was able to oxidize and grow on C 1 –C 3 compounds. Oxidation of ethane did occur simultaneously with methane, while propane consumption only started once methane and ethane became limited. Butane oxidation was not observed. Transcriptome data showed that pmoCAB1 and pmoCAB3 were induced in the absence of methane and the expression of pmoCAB3 increased upon propane addition. Together the results of our study unprecedently show that a pMMO-containing methanotroph is able to co-metabolize other gaseous hydrocarbons, beside methane. Moreover, it expands the substrate spectrum of verrucomicrobial methanotrophs, supporting their high metabolic flexibility and adaptation to the harsh and dynamic conditions in volcanic ecosystems.",
"introduction": "Introduction Methane (CH 4 ) is a powerful greenhouse gas, which is released to the atmosphere from both natural and anthropogenic sources. About 70–80% of CH 4 is generated biologically and a large part of it is removed in the stratosphere and troposphere through reactions with chlorine and ⋅ OH radicals ( Le Mer and Roger, 2001 ). In addition, microbial methane oxidation is an important terrestrial methane sink ( Conrad, 2020 ). Bacteria can convert CH 4 to methanol aerobically using the enzyme methane monooxygenase (MMO; Conrad, 2009 ). Under anaerobic conditions, mostly methanotrophic archaea remove methane via reverse methanogenesis ( Welte et al., 2016 ). After the original discovery by Soehngen (1906) , it was believed for a long time that aerobic methanotrophy was restricted to the phylum Proteobacteria, specifically in the subphyla α- and γ-Proteobacteria ( Op den Camp et al., 2009 ). During the past decade, it was discovered that two bacterial phyla contained new methanotrophic representatives: the intra-aerobic NC10 ( Raghoebarsing et al., 2006 ; Ettwig et al., 2010 ; Hu et al., 2014 ) and the Verrucomicrobia ( Dunfield et al., 2007 ; Pol et al., 2007 ; Islam et al., 2008 ). The phylum Verrucomicrobia includes highly acidophilic and mesophilic Methylacidimicrobium species (optimum pH 1–3: temperature 30–44°C) ( Sharp et al., 2014 ; van Teeseling et al., 2014 ) and thermophilic but less acidophilic strains of the genus Methylacidiphilum (optimum pH 2–2.7; temperature 50–55°C) ( Dunfield et al., 2007 ; Pol et al., 2007 ; Islam et al., 2008 ; Erikstad et al., 2019 ). Methylacidiphilum fumariolicum SolV is the most studied verrucomicrobial methanotroph to date and was initially discovered in the Solfatara volcano near Naples (Italy) ( Pol et al., 2007 ). Strain SolV grows optimally at pH 2.7 and 55°C and it fixes CO 2 via the Calvin cycle and N 2 gas through a nitrogenase enzyme ( Khadem et al., 2010 , 2011 ). Methane can be used as energy source, but strain SolV is also able to use hydrogen gas as substrate, even at sub-atmospheric concentrations ( Mohammadi et al., 2017 ; Schmitz et al., 2020 ). Beside methane, the Solfatara volcano in Naples (and many other volcanic areas) emits a mixture of gas that also includes ethane (C 2 H 6 , 805–1218 ppbv), propane (C 3 H 8 , 68–178 ppbv), and butane (C 4 H 10 , 8–18 ppbv) ( Capaccioni and Mangani, 2001 ). These gases are particularly important because they could serve as additional substrate for microorganisms. Further, they are present, together with methane, in natural gas, which is commonly used in households and industries. The oxidation of C 2 –C 4 compounds is mainly observed in a group of bacteria that includes the genera Corynebacterium , Nocardia , Mycobacterium , Rhodococcus , Pseudomonas and the sulfate-reducing bacteria Desulfosarcina/Desulfococcus and Desulfotomaculum ( Takahashi, 1980 ; Ashraf et al., 1994 ; Hamamura et al., 1999 ; Kinnaman et al., 2007 ; Kniemeyer et al., 2007 ). Recently, oxidation of alkanes under anoxic conditions was reported in archaea and catalyzed by the enzyme ethyl/methyl-coenzyme M reductase (MCR; Laso-Pérez et al., 2016 ; Borrel et al., 2019 ; Chen et al., 2019 ; Seitz et al., 2019 ; Wang et al., 2019 ). In the past, the aerobic oxidation of methane and short-chain alkanes was considered to be carried out by separate groups of microorganisms ( Crombie and Murrell, 2014 ). However, early studies already obtained indications that methanotrophs might be able to oxidize ethane, propane and butane ( Leadbetter and Foster, 1960 ; Hazeu and de Bruyn, 1980 ; Shennan, 2006 ). In 2010, Stable Isotope Probing (SIP) experiments linked the oxidation of ethane to the family Methylococcaceae and the oxidation of propane to unclassified γ-Proteobacteria ( Redmond et al., 2010 ). Methylocella silvestris (α-Proteobacteria) was the first strain that showed simultaneous growth on methane and propane. This strain contained both a soluble methane monooxygenase (sMMO) and a soluble propane monooxygenase (PrMMO) ( Crombie and Murrell, 2014 ). Genes encoding proteins of the methylmalonyl-CoA pathway of propionate oxidation were induced during growth on propane. The enzymes involved in aerobic hydrocarbon oxidation are usually soluble di-iron monooxygenases complexes consisting of multiple associated proteins ( Shennan, 2006 ). sMMO also has this structure and exhibits a larger substrate range than the copper-containing particulate methane monooxygenase (pMMO; Burrows et al., 1984 ). However, the butane monooxygenases of Nocardiodes CF8 and Mycobacterium probably contain copper ( Hamamura and Arp, 2000 ; Coleman et al., 2012 ). The mechanism of hydrocarbon oxidation starts with the conversion of the alkane into an alcohol. More specifically, ethane is oxidized to ethanol, acetaldehyde and acetate; propane can be oxidized at the terminal or subterminal carbon atom, leading to the formation of 1-propanol, propionaldehyde, and propanoic acid in case of terminal oxidation and to 2-propanol and acetone in case of sub-terminal oxidation. Butane, instead, is oxidized to 1-butanol, butyraldehyde, and butyric acid ( Shennan, 2006 ). The genome of strain SolV does not encode sMMO, nor propane or butane monooxygenases, but it shows the presence of three operons for the membrane-bound pMMO. pmoCAB1 and pmoCAB2 operons are located in close proximity in the genome and their PmoA subunits share 84% amino acid identity. The pmoCAB3 operon, instead, is distantly located and its PmoA3 subunit only shares 41% amino acid identity to PmoA1 and PmoA2. Experimental data have demonstrated that the expression of pmoCAB1 and pmoCAB2 is regulated by oxygen concentrations ( Khadem et al., 2012 ), whereas pmoCAB3 expression was so far not detected under any growth condition tested. One hypothesis proposes that pmoCAB3 is of ancestral origin and its function could differ from methane oxidation ( Fuerst, 2014 ). Therefore, the aim of this study was to test the ability of strain SolV to grow on short-chain alkanes and to investigate the expression of the three pmo operons. Here we report that M. fumariolicum SolV can grow on ethane and propane, but not on butane. When methanol is supplied to a SolV culture with no oxygen limitation, expression of pmoCAB1 and pmoCAB3 could be detected. Furthermore, pmoCAB3 expression increased upon propane addition.",
"discussion": "Discussion In this study, we show that M. fumariolicum SolV is able to co-metabolize ethane and propane with methane or methanol, but not butane. To our knowledge, the ability to use other gaseous hydrocarbons for growth is unprecedented in a pMMO-encoding methanotroph (with no sMMO present). Most methanotrophs are highly selective, to the point that they only grow on methane and its one-carbon derivatives such as methanol, and cannot grow on complex, multi-carbon substrates like sugars or organic acids. However, the facultative methanotroph Methylocella silvestris that only contains sMMO (and PrMMO) is able to grow on acetate, ethanol, pyruvate, succinate, malate and propane in addition to methane and methanol ( Dedysh et al., 2005 ; Chen et al., 2010 ; Crombie and Murrell, 2014 ). In 2010, Redmond and colleagues ( Redmond et al., 2010 ) suggested an alternative function for pmoA genes, based on the ability of some pmoA -containing microorganisms to incorporate carbon derived from the oxidation of ethane and propane. Our study demonstrates that pMMO is indeed involved in the consumption of alkanes in strain SolV. Further, we could show that without oxygen limitation and in the presence of ethane/propane and methanol, pmoCAB1 and pmoCAB3 were expressed. This is the first time that pmoCAB3 expression in SolV is ever detected. Previous work only documented the alternate expression of pmoCAB1 and pmoCAB2 in relation to oxygen ( Khadem et al., 2012 ). The increased expression of pmoCAB3 upon propane addition suggests that these genes could be involved in alkane oxidation. At the current state we cannot conclude which pMMO enzyme complex performs ethane and propane oxidation nor we can exclude that it is a combination of multiple enzymes. Further experiments, including more detailed and time-resolved RNAseq studies and expression of this operon in an alternative host, need to be performed to elucidate the exact role of pmoCAB3 in alkane consumption. It is expected that ethane and propane are first converted by pMMO to ethanol and propanol, respectively. The XoxF-type PQQ-dependent methanol dehydrogenase of strain SolV was shown to convert these alcohol to their aldehyde (acetaldehyde, propanaldehyde) and acid (acetate, propanoate) forms ( Pol et al., 2014 ). The transcriptome data did not reveal upregulation of genes that could point to the further oxidation of these metabolites. Acetate could be shuttled into the central metabolism by the acetyl-CoA synthetase present in the genome of strain SolV (Mfumv2_2288). The enzymology of propane oxidation after the first two steps is poorly understood. Crombie and Murrell (2014) found an induction of the methylmalonyl-CoA pathway enzymes during growth on propane. However, the genome of strain SolV does not encode for these enzymes. However, acetyl-CoA synthetase (EC 6.2.1.1) is also know to convert propanoate into propanoyl-CoA. Metabolomic studies could shed light on the pathways involved. The oxidation of ethane and propane in M. fumariolicum SolV proceeded at different rates. In particular, ethane seemed to be preferred over propane since (i) it could be oxidized simultaneously with methane, (ii) the oxidation rate was faster, and (iii) it led to a higher increase in biomass (70% vs 34%). This probably depends on the ability of pMMO of binding and converting molecules with different number of carbon atoms. Moreover, differences in the ethane oxidation rate could be noticed in different conditions. In particular, a 10-fold increase in the ethane consumption rate was calculated in the continuous culture compared to the batch incubations. This discrepancy could be due to CO 2 or NH 4 + limitations in the batch experiments. The solubility of the different alkanes at 55°C do not differ much. The values calculated from mole fractions taken from Wilhelm et al. (1977) were 0.92 mM for methane, 1.04 mM for ethane, 0.79 mM for propane, and 0.56 mM for butane. Contrary to ethane and propane, butane consumption was not observed in strain SolV. Butane oxidation, together with propane and ethane, was detected in Methylosinus trichosporium OB3b. This bacterium encodes both sMMO and pMMO ( Burrows et al., 1984 ), but its pMMO is different than the ones encoded by strain SolV. PmoA from M. trichosporium OB3b only shares 53% amino acid identity to strain SolV’s PmoA1, 51% to PmoA2 and 40% to PmoA3. The sMMO-containing methanotroph Methylococcus capsulatus Bath also showed the ability of oxidizing C 1 –C 8 compounds ( Colby et al., 1977 ). The oxidation of these alkanes in M. capsulatus and M. trichosporium , however, was not linked to growth. Additionally, butane oxidation has been documented in the genera Nocardioides, Mycobacterium, Giesbergeria, Ramlibacter, Arthrobacter, Brevibacterium ( McLee et al., 1972 ; Hamamura et al., 1999 ; Hamamura and Arp, 2000 ; Deng et al., 2018 ) and in the β-proteobacterium Thauera butanovora ( Arp, 1999 ). The butane monooxygenase of T. butanovora (sBMO) presents high identity (38–65%) to sMMO as it contains three subunits α, β and γ encoded by bmoX , bmoY and bmoZ genes and a non-haem carboxylate-bridged diiron site ( Sluis et al., 2002 ). The sBMO has a much lower affinity for methane (1.1 mM) compared to sMMO (3–13 μM) ( Cooley et al., 2009 ). The butane degrading strain CF8, instead, seems to possess a pBMO similar to pMMO with subunit identities of 34–47% ( Hamamura et al., 1999 ; Kinnaman et al., 2007 ; Sayavedra-Soto et al., 2011 ). A copper containing monooxygenase in Mycobacterium able to oxidize C 2 –C 4 alkanes was also described ( Coleman et al., 2012 ). In conclusion, this study demonstrates that, beside methane and hydrogen, verrucomicrobial methanotrophs are also able to co-metabolize higher alkanes. This result is particularly important in view of the ecological role of these bacteria in the environment. Methanotrophic Verrucomicrobia appear to be not only extremely resistant to thermoacidic geothermal volcanoes, but also remarkably flexible in terms of substrate utilization. Their metabolic flexibility regarding carbon compounds could be partly provided by the differential expression of the pmoCAB copies in relation to the substrate available."
} | 3,604 |
37421944 | null | s2 | 6,138 | {
"abstract": "Engineering synthetic heterotrophy is a key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades-yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are a key to obtaining highest growth rates and yields with minimal metabolic engineering effort. Concurrently, results indicate that \"extrinsic\" factors-specifically, upstream genes that direct flux of pentoses into central carbon metabolism-are rate-limiting. We also reveal that yeast metabolism is innately highly adaptable to rapid growth on non-native substrates and that systems metabolic engineering (i.e., functional genomics, network modeling, etc.) is largely unnecessary. Overall, this work provides an alternate, novel, holistic (and yet minimalistic) approach based on integrating non-native metabolic genes with a native regulon system."
} | 277 |
38846575 | PMC11153752 | pmc | 6,139 | {
"abstract": "The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.",
"introduction": "1 Introduction Rhizosphere microbes have co-evolved with host plants and usually form mutually beneficial relationships. In return for carbon in the form of root exudates ( Bais et al., 2006 ; De Deyn et al., 2008 ; Hu et al., 2018 ; Vives-Peris et al., 2020 ), rhizosphere microbes perform a range of functions that benefit their host plants. These encompass the conversion of essential nutrients (e.g., nitrogen, phosphate, zinc, iron) into more accessible forms for plant assimilation ( Tariq et al., 2007 ; Sharma et al., 2013 ; Kuan et al., 2016 ; Satyaprakash et al., 2017 ; Pérez-Izquierdo et al., 2019 ), conferring resilience against environmental stressors such as pathogen infections and water limitations ( Rodriguez et al., 2008 ; Mendes et al., 2011 ; Bulgarelli et al., 2013 ; Bhattacharyya et al., 2021 ), secretion of plant growth promoting hormones ( van der Lelie et al., 2009 ; Bulgarelli et al., 2013 ) among others. Given their high abundance, diversity and activity, rhizosphere microbes are often considered as “the second genome of plants” ( Berendsen et al., 2012 ). To advance our understanding and gain more insight into rhizosphere assembly processes, it is critical to recover and to recreate a representative rhizosphere community that accurately encapsulates the inherent diversity and functions of the rhizobiome for detailed lab investigations. Generally, representative reduced complexity consortia (RCC) are constructed employing bottom-up and top-down approaches. Bottom-up method focuses on building synthetic communities from cultivated isolates. However, this method could inadvertently exclude microbes that resist cultivation under routine cultivation and incubation conditions including some rare and dormant taxa ( de Souza et al., 2020 ; McClure et al., 2020 ; Choi et al., 2021 ). In addition, for subsequent consortia stability, knowledge about individual microbes and their interactions with partner microbes is imperative ( Kaeberlein et al., 2002 ; Gilmore et al., 2019 ; Wu et al., 2020 ). Conversely, the top-down approach employs enrichments to generate consortia of reduced complexity after multiple passages ( Gilmore et al., 2019 ; Díaz-García et al., 2021 ). However, this approach may only yield fast-growing generalists of low diversity if routine carbon sources are used, overlooking the true rich phylogenetic and functional diversity of rhizosphere ( Kaeberlein et al., 2002 ; Wu et al., 2020 ). Consequently, vital to construction of useful RCC is the design framework, that demonstrates stability, complexity, reproducibility, scalability, and genetic tractability—which appears to be generally lacking ( Wu et al., 2020 ; Liang et al., 2022 ). In this study, we developed a systematic and standardized framework to generate RCC that optimally represent the rhizosphere microbiome of Brachypodium distachyon and satisfies all the above criteria. Previously, we grew young Brachypodium distachyon using natural soil in standardized fabricated ecosystems (EcoFABs; Zengler et al., 2019 ) as well as conventional pots and tubes under controlled conditions, and demonstrated that the rhizosphere microbiome of Brachypodium was clearly distinct from bulk soil microbiome irrespective of the growth container ( Acharya et al., 2023 ). Subsequently, in this current study, using the naturally selected co-localized root microbiome as inoculum, we performed a large number of high-throughput enrichments to derive rhizobiome relevant RCC. To do so, we considered critical parameters such as choice of carbon substrate, differential microbial growth rates and even root-enriched inoculum from different growth containers. We measured the diversity and richness of the developing communities across multiple generations, identified the key microbial taxa that were significantly influenced by these factors, and discerned the keystone taxa that controls the interactions within the core community members. Furthermore, we demonstrated the validity of our framework showing stability, tractability, reproducibility, and revivability from both original enrichments and preserved glycerol stocks, all key factors important for effective use and application of microbial consortia for field studies. Our approach of generating RCC using this robust and standardized framework that considers co-localization, and microbial interactions is a significant advance toward microbial-based solutions for agriculture and ecosystem management toward climate change mitigation strategies.",
"discussion": "4 Discussion A key innovation of our approach to enrichment of reduced complexity consortia from the rhizosphere is the multiplexed design of crucial parameters. This includes selection of naturally co-localized root microbes as inocula, selecting root exudate-mimicking carbon substrates, and implementing transfer intervals to allow both slow and fast-growing microbes. Moreover, we included Brachypodium distachyon grown in various containers to account for the potential impact of different growth chambers on rhizobiome interactions ( Yee et al., 2021 ). Generally, microbial communities from EcoFAB were distinct from those sourced from pots and tubes ( Supplementary Figure S2 ). This disparity may stem from inherent differences between EcoFAB and other containers due to space footprint or humidity control. These variations could directly influence root architecture ( Yee et al., 2021 ) and root exudate composition and patterns, ultimately impacting the rhizobiome. Besides, previous study demonstrated the significant effect of inoculum on consortium richness ( Zegeye et al., 2019 ). Our study further emphasizes the importance of inoculum composition as a critical factor when constructing reduced complexity microbial consortia. Different root exudate compounds specifically favored the growth of certain ASVs, particularly those known for promoting plant growth. Asparagine, the dominant root exudate from Brachypodium distachyon ( Kawasaki et al., 2016 ), significantly enriched ASVs from genera Bradyrhizobium and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium ( Supplementary Table S5 ). Both Bradyrhizobium and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium are known to be associated with root nodules promoting plant growth ( Mangeot-Peter et al., 2020 ; Jaiswal et al., 2021 ). Mycobacterium and Delftia were significantly enriched with glucose, both genera have been known to promote plant growth by solubilizing phosphate or fix nitrogen as non-rhizobial endophytes ( Hakim et al., 2021 ; Zhou et al., 2021 ). Brevibacillus , significantly enriched in glutamine-amended samples, have been known previously to promote plant growth by inhibiting pathogenic microbes ( Sheng et al., 2020 ). Interestingly, glucuronic acid significantly increased the diversity of ASVs enriched when inocula was derived from pots and enriched the second-highest number of distinct ASVs regarding different carbon substrates ( Figures 1 , 2 ). Although the general impact of glucuronic acids on the rhizosphere is relatively unexplored, recent studies indicate that glucuronic acid positively correlates with rhizosphere bacteria and is considered a key metabolite ( Baker et al., 2022 ). Taken together, these results support that plants secrete diverse compounds through root exudates to selectively enrich for microbes with specific plant growth promoting traits. Therefore, our findings emphasize the importance of using relevant carbon substrates when attempting to enrich microbial consortia of varied phylogenies and functions from rhizosphere. Moreover, choice of carbon substrates can distinctly influence the growth of fast- vs. slow-growing microbes. While the rhizobiome is typically believed to be dominated by fast-growing copiotrophs ( Ling et al., 2022 ), slow-growing oligotrophs still play crucial roles including protection against plant diseases ( van der Voort et al., 2016 ) and combating environmental stress ( Hartman et al., 2018 ). Additionally, many slow-growing microbes are less abundant in the rhizosphere, making direct isolation and characterization challenging without intelligent enrichment efforts ( Delmont et al., 2015 ). Our results show that diverse carbon substrates enriched similar fast-growing microbes ( Figure 3C ). This could be attributed to the fact that the original inocula originating from immature roots, are often dominated by fast-growing microbes ( De Leij et al., 1995 ; Nacamulli et al., 2006 ). To note, glucose and malate, which have relatively simple chemical structures, can significantly enrich more of fast-growing microbes compared to slow-growing microbes ( Figures 3B , C ). However, distinct differences are observed in the enrichment potential of various carbon substrates for slow-growing microbes ( Figure 3B ). Notably, 1/10 R2A substantially enriched slow-growing microbes, including the phyla Acidobacteria and Verrucomicrobia, which are often recognized as difficult to enrich microbes ( Sangwan et al., 2005 ; Ward et al., 2009 ) but with high potential for promoting plant growth ( Kielak et al., 2016 ; Bünger et al., 2020 ). Interestingly, another complex carbon source (mixed carbon) does not significantly enrich slow-growing microbes as one might expect ( Figures 3A , B ). We think this observation could be attributed to several factors. First, R2A medium and its diluted version contain a broader range of metabolites compared to our mixed carbon substrates ( de Raad et al., 2022 ), which could be crucial for enriching slow-growers, given their reliance on specific substrates for survival in the rhizosphere ( Zhalnina et al., 2018 ). Second, the total organic carbon (TOC) concentration in 1/10 R2A is lower than in mixed carbon enrichment ( Supplementary Table S2 ), potentially favoring oligotrophs that thrive in nutrient-limited environments. Third, we utilized consistent concentrations of different root exudates for our experiments, while in natural settings, these concentrations could vary substantially ( Kawasaki et al., 2016 ), which might directly impact the enrichment of diverse microbes. These observations further underscore the critical role of selecting diverse and complex carbon sources to effectively enrich rare, yet essential, slow-growing microbes within the rhizosphere ( Wu et al., 2020 ). Additionally, fast- and slow-growing consortia showed stark differences in the keystone taxa ( Figure 4 ), which are crucial nodes that hold central positions based on highest eigenvector centrality ( Peschel et al., 2021 ). A node with high eigenvector means it connects to numerous central nodes, making it the influential node in the network analysis ( Zhang, 2009 ). Consequently, keystone taxa can be considered ecologically important microbes responsible for shaping the microbial community structure and dynamics ( Agler et al., 2016 ). Notably, 8 keystone taxa from the hubs belong to genera known to exhibit PGP traits ( Kielak et al., 2016 ; Alijani et al., 2020 ; Jaiswal et al., 2021 ; Sah et al., 2021 ; Juma et al., 2022 ; Platamone et al., 2023 ). The robust link between keystone taxa and PGP traits underscores the significant impact of PGP bacteria on the dynamics of the rhizosphere microbiome ( Pii et al., 2015 ), stressing the need for careful selection in constructing microbial consortia. Despite most keystone taxa displaying low (<0.5%) abundance across various generations under consistent conditions, they are persistent through different generations ( Supplementary Table S8 ). Notably, specific keystone taxa, such as the Rhizobiaceae family and Terriglobus genus, remain constant across comparisons between fast and slow growers ( Figure 4 ), as well as across different generations ( Supplementary Figure S9 ). This underscores the vital role and involvement of keystone species, especially slow-growing microbes such as genus Terriglobus , in influencing rhizosphere dynamics, highlighting the necessity of incorporating such microbes into rhizosphere microbial consortia design and construction. We evaluated the efficacy of our pipeline by assessing the stability and diversity of our enrichments over multiple generations ( Supplementary Figure S1 ). We tested these two parameters because stability and tractability are two essential features for creating successful microbial consortia ( Zegeye et al., 2019 ; McClure et al., 2020 ; Shayanthan et al., 2022 ). We found a significant reduction in species richness in our RCC-enriched samples across all generations ( Supplementary Figure S4 ) from original inocula. Stability assessments across generations (1, 3, 6, and 9) demonstrated consistent low diversity (<70 observed species), and high consistency in species composition, regardless of the variables tested, highlighting the enriched samples’ stability ( Figure 5 ; Supplementary Figure S5 ). This suggests that our reduced-complexity microbial communities likely achieved stability at early generations, except for those amended with 1/10 R2A medium, which showed increased diversity. Nonetheless, network analysis of 1/10 R2A samples across generations revealed consistent clustering ( p- values < 0.001), indicating similar community structures despite the diversity increase (as shown in Supplementary Figure S9 ). These results affirm the effectiveness of our framework in developing stable and traceable microbial consortia. As a last but critical step, we tested a subset of 5 of our microbial consortia that had been preserved in glycerol stocks to further assess their reproducibility, stability, and revivability after several months. Consistent revival and propagation from glycerol stocks is also a highly desired trait for microbial consortia to enable study across multiple labs and timescales and eventually for agricultural applications. Generally, our RCC exhibited consistent genera (in respect to both presence/absence and relative abundances, Figure 6 ), richness, and evenness across different transfers after revival ( Supplementary Figure S6 ) and have tractable numbers (18 to 44) of observed OTUs, demonstrating good reproducibility. The low values from the volatility tests between transfers ( Supplementary Figure S7 ) also support the stability of the derived microbial consortia. We also verified the revivability of our derived consortia, showing that the communities revived from glycerol stocks have a similar microbial composition to the original culture before freezing ( Supplementary Table S6 ). Even though the glycerol stocks contained some dead cells, we upscaled the volume to 100 mL by serial transfers and subsequently centrifuged 80 mL of this to extract DNA. This process ensures that the sequencing results predominantly reflect the DNA from viable cells. Furthermore, our flow cytometry evaluation of the initial glycerol stocks and their serial transfers consistently showed a stable and substantial number of active cells ( Supplementary Table S10 ). Network analysis revealed consistent patterns over different transfer periods and shared key taxa, including genera known for PGP traits ( Nacamulli et al., 2006 ; Menéndez et al., 2020 ). Yet, it should be noted that each individual node may display varying likelihoods of interaction with other members within the same community when varying time intervals ( Supplementary Figure S8 ). For instance, a node from the genus Terriglobus , which is known for slow growth rates ( Kalam et al., 2020 ), demonstrates more active interaction with other nodes over 7-day intervals as denoted by the thickness of the edges. On the other hand, nodes from the Cohnella and Chitinophaga genera, recognized as fast-growing microbes ( McKee and Brumer, 2015 ; Johnson et al., 2021 ), exhibited increased interactions with other nodes over 3-day intervals. These observations underscore the importance of longer incubation times when attempting to construct consortia, to ensure the inclusion of more slow-growing microbial members. In summary, we developed a systematic high-throughput schema to enrich co-located microbes from the rhizosphere of Brachypodium distachyon , encompassing key factors like root exudates, inoculum source, and transfer duration. Our findings underscore the impact of the initial inoculum and carbon sources in shaping microbial community composition. We further highlight the influence of plant growth container type on enrichment outcomes, and our results demonstrate that diverse carbon sources similar to root exudates enhance specific beneficial microbes. We discovered that keystone taxa varied between fast- and slow-growing enriched communities and across different generations, however, they belong to microbes with plant-growth-promoting traits. Overall, our study represents a significant stride toward developing a framework for assembling stable, rhizosphere consortia based on microbial colocalization and interaction for agricultural and environmental enhancements."
} | 4,756 |
32009675 | PMC6959382 | pmc | 6,140 | {
"abstract": "We consider systems of agents interacting through topological interactions. These have been shown to play an important part in animal and human behavior. Precisely, the system consists of a finite number of particles characterized by their positions and velocities. At random times a randomly chosen particle, the follower, adopts the velocity of its closest neighbor, the leader. We study the limit of a system size going to infinity and, under the assumption of propagation of chaos, show that the limit kinetic equation is a non-standard spatial diffusion equation for the particle distribution function. We also study the case wherein the particles interact with their K closest neighbors and show that the corresponding kinetic equation is the same. Finally, we prove that these models can be seen as a singular limit of the smooth rank-based model previously studied in Blanchet and Degond (J Stat Phys 163:41–60, 2016 ). The proofs are based on a combinatorial interpretation of the rank as well as some concentration of measure arguments.",
"conclusion": "Conclusion In this paper, we have put forward a particle interaction model where particles interact with their nearest neighbor. We have shown that the large particle limit under the Propagation of Chaos assumption is a spatial diffusion for the particle distribution function corrected by an anti-diffusion term acting on the spatial density. We have shown that the appearance of this anti-diffusion term depending on the spatial density results from the fact that the interactions are mass-preserving. We have also considered a model in which particles interact with their K nearest neighbors, for a fixed value of K , showing that the corresponding kinetic model is the same as in the nearest neighbor interaction case. Finally, we have linked this work with the previous article [ 10 ] where smooth rank-based dynamics were considered and shown that the kinetic nearest-neighbor model can be recovered from the former through a singular limit involving a scaling of the interaction kernel. The kinetic models obtained here, as in [ 10 ], are novel. Their mathematical theory is entirely open: proving existence and uniqueness of solutions, investigating large-time behavior, equilibria and other qualitative properties of the solutions will require the establishment of an appropriate mathematical framework. In parallel, more elaborate physical interaction models (such as the Cucker–Smale [ 20 ] or Motsch–Tadmor [ 42 ] models) should also be considered. One important question is to investigate how the present results are robust to the introduction of noise in the interaction dynamics. Indeed, noise play an important part of many flocking models in nature. Finally, adequate numerical methods for the kinetic models must be developed and the assessment of the kinetic models against the particle ones in realistic situations should be carefully documented so that these models can be used in practice.",
"introduction": "Introduction In the literature on animal behavior including fish [ 3 ], birds [ 40 ] and even pedestrians [ 34 ], interactions between individuals are often assumed to be strongly dependent on their relative distance. However it has recently been demonstrated that individuals in bird flocks interact with their nearest neighbors irrespective of their distance [ 5 , 18 ]. More precisely, the authors in [ 5 ] claim that each bird interacts with between six to eight of its closest neighbors. The authors coined the term of “topological interaction” to refer to such interaction mechanisms and ”topological distance” to refer to how many other individuals were closer. Even though the reality of this topological interaction has been debated [ 26 ], it now seems to receive consensus following reports that self-propelled particle models based on topological interactions successfully reproduce the observed experimental features [ 11 , 14 , 30 ]. The understanding that birds interact through topological rather than metric distance has generated an intense literature. Topological interactions have been introduced to the modeling of many natural phenomena, from birds [ 35 ] to pedestrians [ 38 ]. Mixed metric-topologic interactions have also been proposed [ 43 , 46 ]. Proof of flocking under topological interaction has been given in [ 33 , 41 , 48 ], while speed to consensus has been shown to depend on the number of interacting neighbors in [ 47 ]. Similarly, interactions depending of the behavior of the closest neighbor are probably at play in human interactions such as portfolio theory [ 6 , 27 , 37 ], competition between coworkers within a firm, risk-taking among traders or aggressive behavior to reach a sexual partner, see for example [ 24 , 36 ]. Rank-based dynamics bears similarities with rearrangement (see e.g. [ 13 ] and references therein). One of the striking features of topological compared to metric interactions is their scale-invariance property. Indeed, irrespective of the bird concentration within the flock, [ 5 ] proved that the interaction features remain unchanged. In human cognition models, it is also more relevant to restrict interactions to the closest neighbors, as the attention of a subject is intrinsically directed to only a few people around him/her [ 44 ]. Interactions with close neighbors do not preclude interactions at a longer range, as interactions spread with the conscious or unconscious signals sent by the subjects in response to these interactions. However, issues such as quantify the propagation speed of information sent via topological interactions are poorly understood so far, in particular in the presence of a large number of subjects. This calls for a large-scale theory of topological interactions, or in other words, for the development of meso or macroscopic models of particle systems connected through topological interactions. The aim of this article is specifically to derive a macroscopic model for a large population of particles interacting through topological interactions, starting from a simple microscopic model. To the best of our knowledge, the present work and its predecessor [ 10 ] are the first to develop a rigorous coarse-graining of topological interactions, with the exception of [ 33 ] which tackled a similar question but for a different kind of interaction, closer to mean-field type interactions. More precisely, we will consider a system with interacting mobile agents. At Poisson random times a given agent selects a partner to interact with according to a probability rule which depends on the proximity rank of the partner. The interaction rule is then very simple: the agent changes its velocity to align with that of its partner. The goal of the present work is, by letting the number of agents tend to infinity, to derive an equation for the probability distribution of the agents in phase space (positions, velocities). Thanks to the choice of this simple interaction rule, inspired from earlier work [ 15 , 16 ], we can concentrate on the mathematical aspects of this derivation. In previous work by the authors [ 10 ], the probability rule solely depended on the proximity rank normalized by the total number of interacting partners (or equivalently, on a proximity rank expressed as a percentage). This rule made the number of potential interacting partners tend to infinity as the number of agents also did so. We will refer to this rule as the ”smooth rank-based dynamics”. By contrast, in the present work, we will concentrate on the case where there is only one interaction partner (the nearest one) or a finite number of them (the K nearest ones), even in the limit of the number of agents tending to infinity. We will refer to these dynamics as the “nearest-neighbor” or “ K -nearest-neighbor” dynamics. In this paper, we show (under the Propagation of Chaos assumption) that the kinetic equation resulting from the nearest-neighbor or K -nearest neighbor dynamics is a nonlinear spatial diffusion equation for the particle distribution function in phase space (position, velocity). This equation has a non-classical feature as it involves a spatial anti-diffusion of the density (which is a velocity integral of the distribution function). We will show that this term results from the constraint that the density must satisfy the continuity equation, a constraint resulting from the preservation of the number of particles in the course of an interaction. By contrast, in the previous work [ 10 ] relative to the smooth rank-based dynamics, we showed (also under the Propagation of Chaos assumption), that the resulting kinetic equation involved a spatially non-local integral equation and that the continuity equation for the density was also satisfied. In the present paper, we also show that we can pass from the non-local integral equation issued from the smooth rank-based dynamics to the nonlinear spatial diffusion equation for the nearest-neighbor dynamics by a process involving a singular concentration of the kernel of the integral equation. This provides a vision of the nearest-neighbor dynamics as a singular limit of the previously studied smooth rank-based dynamics. Rank-based dynamics (either smooth or nearest-neighbor) are natural from a mathematical point of view. The proximity rank includes information about the most immediate interaction partners of a given particle. Although the rank is a highly non-linear function of the particle positions and is subject to jumps when two particles cross, it has robust properties such as invariance by permutation of the particle numbers, and has combinatorial interpretation: the probability for an agent to have a rank k with respect to agent i is equal to the probability of having \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$k-1$$\\end{document} k - 1 agents between them. Our results strongly rely on this interpretation of the rank, together with some concentration of measure arguments. Rank-based dynamics also exhibit a certain universality, as their kinetic model does not depend on the (finite) number of interacting agents. The kinetic equation that we derive in this work leads to many questions. This equation is a non-standard diffusion equation with mainly unkonwn properties, from the perspective of well-posedness, large-time behavior, regularity, etc. We believe that these questions open fascinating new directions of research in kinetic theory. Kinetic models of flocking or swarming behavior have been widely investigated in the context of metric interactions. The literature is vast and it is virtually impossible to be exhaustive. Below is a sample of major publications on this topic. Derivation of kinetic models from underlying particle models have been established in [ 12 , 23 , 32 , 45 ]. Flocking behavior and pattern formation has been investigated in [ 1 , 9 , 17 , 31 , 32 , 42 ]. Equilibria and phase transitions in kinetic flocking models have been studied in [ 7 , 21 , 22 ]. Passage from kinetic to hydrodynamic descriptions of flocking has been investigated in [ 8 , 21 – 23 , 25 , 28 , 39 ]. Numerical simulation methods have been put forward in [ 2 , 29 ]. The organization of the paper is as follows. Section 2 is dedicated to the presentation of the models and our main results, as well as a detailed discussion of them. Section 3 covers the nearest-neighbor case and the derivation of the kinetic model in this case. Section 4 extends these results to the case where the particles interact with their K closest neighbors. Section 5 develops the proof that the kinetic model of the nearest-neighbor (or K -nearest-neighbor) interaction is the limit of the kinetic smooth rank-based interaction model of [ 10 ]. A conclusion to this article is given in Sect. 6 .",
"discussion": "Discussion As mentioned in Sects. 2.2 and 2.3 , the kinetic models derived from the nearest neighbor interaction or the K -nearest neighbor interactions are the same. On the other hand, these models differ quite significantly from that obtained from the smooth ranked-based dynamics of [ 10 ] as recalled in Sect. 2.4 . However, we show in Sect. 5 that the former are limits of the latter when the interaction kernel K concentrates (with a convenient scaling) near zero. In this limit, since only the closest neighbors interact, and these closest neighbors are likely to be spatially close (especially when the density is large) the spatially non-local integral operator appearing in ( 6 ) converges to the diffusion operator ( 4 ). Note that this diffusion is multiplied by an inverse power of the density. This is easily understood as, when the density is small, the particles are very far apart, resulting in spatial communications between the particles over larger distances, and eventually, into a larger diffusion coefficient. This interpretation is reinforced by the fact that the inverse power of the density depends on the dimension, in the same way as the scaling between the inter-particle distance and the density depends on the dimension. In [ 10 ], we noted that any solution of the smooth rank-based dynamic kinetic model ( 6 ) satisfies the mass conservation equation \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} \\partial _t \\rho + \\nabla _x \\cdot (\\rho u) = 0, \\end{aligned}$$\\end{document} ∂ t ρ + ∇ x · ( ρ u ) = 0 , with \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\rho = \\int f \\, \\;\\mathrm{d}v$$\\end{document} ρ = ∫ f d v and \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\rho u = \\int f \\, v \\;\\mathrm{d}v$$\\end{document} ρ u = ∫ f v d v . The nearest-neighbor kinetic model ( 3 ) also satisfies the mass conservation equation. To see this, it is enough to show that \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\int Q(f) \\, \\;\\mathrm{d}v = 0$$\\end{document} ∫ Q ( f ) d v = 0 . But we easily check that it is the case. Indeed: \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} \\int Q(f) \\, \\;\\mathrm{d}v= & {} \\frac{1}{\\rho ^{\\frac{2}{d}}} \\left[ \\Delta _x \\left( \\int f \\, \\;\\mathrm{d}v \\right) - \\frac{1}{\\rho } \\left( \\int f \\, \\;\\mathrm{d}v \\right) \\Delta _x \\rho \\right] \\\\= & {} \\frac{1}{\\rho ^{\\frac{2}{d}}} \\left[ \\Delta _x \\rho - \\Delta _x \\rho \\right] = 0. \\end{aligned}$$\\end{document} ∫ Q ( f ) d v = 1 ρ 2 d Δ x ∫ f d v - 1 ρ ∫ f d v Δ x ρ = 1 ρ 2 d Δ x ρ - Δ x ρ = 0 . The collision operator ( 4 ) has the form of a spatial diffusion of f but with an anti-diffusion in \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\rho $$\\end{document} ρ . In fact, this anti-diffusion is exactly the term that needs to be added to turn a pure spatial diffusion \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\rho ^{-\\frac{2}{d}}\\Delta _x f$$\\end{document} ρ - 2 d Δ x f into an operator that conserves mass i.e. that satisfies \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\int Q(f) \\, \\;\\mathrm{d}v = 0$$\\end{document} ∫ Q ( f ) d v = 0 . At the microscopic level, the collision dynamics describes particles communicating their velocity to spatially distant (although close) neighbors. Therefore, information about the velocity distribution propagates to neighboring particles randomly leading to a spatial diffusion of this distribution. However, this spatial diffusion of the velocity distribution is constrained to obey local mass conservation, which is the reason of the anti-diffusion term acting on the density, as stressed above. If there is no spatial variation of the velocity distribution, particles communicating their velocity to their neighbors will not modify the velocity distribution in this neighborhood, which explains why the collision operator vanishes in this case. The well-posedness theory of ( 3 ) is still open but from this remark we can conjecture that the model is well-posed. Indeed, apart from a mass-carrying component, the equation is a spatial diffusion. And the mass carrying component satisfies a continuity equation. So, it seems that the model couples two components each of which solves a well-posed equation. Of course, the coupling is non-trivial and this may result into a lack of well-posedness. This issue will be dealt with in future work."
} | 4,539 |
24937478 | PMC4061089 | pmc | 6,141 | {
"abstract": "Apical lesions on Porites astreoides were characterized by the appearance of a thin yellow band, which was preceded by bleaching of the coral tissues and followed by a completely denuded coral skeleton, which often harbored secondary macroalgal colonizers. These characteristics have not been previously described in Porites and do not match common Caribbean coral diseases. The lesions were observed only in warmer months and at shallow depths on the fore reef in Belize. Analysis of the microbial community composition based on the V4 hypervariable region of 16S ribosomal RNA genes revealed that the surface microbiomes associated with nonsymptomatic corals were dominated by the members of the genus Endozoicomonas , consistent with other studies. Comparison of the microbiomes of nonsymptomatic and lesioned coral colonies sampled in July and September revealed two distinct groups, inconsistently related to the disease state of the coral, but showing some temporal signal. The loss of Endozoicomonas was characteristic of lesioned corals, which also harbored potential opportunistic pathogens such as Alternaria, Stenotrophomonas , and Achromobacter . The presence of lesions in P. astreoides coincided with a decrease in the relative abundance of Endozoicomonas , rather than the appearance of specific pathogenic taxa.",
"introduction": "Introduction Healthy corals are crucial to the productivity and sustainability of reef ecosystems and surrounding human communities [1] . However, a decline in coral reefs has been documented over the last decades. Global climate change, increasing ocean acidification, overfishing and other human activities have all been linked to a decrease in coral cover world-wide and/or a rapid structural change, often associated with the loss of biodiversity in reef ecosystems [2] – [7] . Even though some coral recovery occurs on impacted reefs, shorter-lived, brooding mounding corals, and especially non-branching members of the Porites genus tend to be over-represented in reefs that recovered from disturbances [2] , [5] , [7] – [9] . This observation led some authors to hypothesize that non-branching Porites spp. will be over-represented on coral reefs in the future [4] , [6] , [8] . This hypothesis, however, assumes that Porites spp. are resilient to other biotic and abiotic stressors. In addition to bleaching (loss or expulsion of symbiotic Symbiodinium spp. dinoflagellates) and decreased calcification rates, global climate change is also associated with an increase in coral diseases (as reviewed by [10] ). There are at least eighteen generally recognized diseases of corals, with at least four pathologies of Porites spp. [11] – [17] . In diseased Porites spp., symptoms can be fairly general, making assignment of gross lesion morphology difficult between diseases. The fulfillment of Koch's postulates linked three closely related strains of Vibrio to the ulcerative white spot disease of P. cylindrica \n [13] , but causes of other observed abnormalities remain elusive. While agents responsible for some of the observed etiologies have been identified and Koch's postulates fulfilled either directly (reviewed by [12] , [15] ) or through the use of host-specific phages [18] – [21] , it is likely that some coral diseases are not caused by specific pathogens. It is hypothesized that many coral diseases are polymicrobial, in which a collection of generic symptoms is elicited by a number of opportunistic pathogens that attack corals when their defenses are compromised or their native microbiota is destabilized [22] , [23] . There is growing evidence that some abnormalities described as coral diseases are associated with general disturbances in the native microbial communities [24] – [26] . It is possible that under some conditions, members of the coral commensal microbiota escape restrictions imposed on them by the host or other members of the host microbiota and start to degrade host tissues [23] , [27] . This hypothesis is supported by two recent reports, in which microbial communities associated with the Yellow Band Disease were not significantly different from the microbial communities of the visually asymptomatic corals [26] , [28] . Addressing these hypotheses is complicated by multiple factors, one of which is the observation that coral-associated microbial assemblages appear diverse, with no clear indication of which members of the microbiome are tightly co-evolved partners and which are commensals or potential opportunistic pathogens [27] . In brooding corals, such as P. astreoides , which vertically transmit bacteria to their larvae, members of microbiomes associated with the same coral appear to be conserved across geographic and temporal scales [29] – [32] . Specifically, studies with clonal libraries and different high-throughput sequencing approaches reported Oceanospirillales, in particular members of the genus Endozoicomonas, as dominant in P. astreoides adult colonies and in larvae at later developmental stages [29] – [32] . Members of the Rhodobacterales, Alteromonadales, Rhizobiales, and Cyanobacteria also appear to be a part of the normal microbiota of P. astreoides \n [29] – [32] . The characterization of the commensal microbiota of P. astreoides makes studies of the diseases of this coral more straightforward. Lesions with characteristics not previously recorded in Porites were observed on P. astreoides at the fore reef in Belize. The lesions were observed in warmer months of 2012 during two independent samplings, and severe damage to the lesioned corals was observed on the second data collection, as well as in cooler months following the data collection. Here, we characterized the surface microbiota from separate colonies of corals exhibiting lesions and from nonsymptomatic corals to determine if the appearance of atypical lesions coincided with a shift in the composition of the microbial community.",
"discussion": "Discussion This study characterizes the surface microbiomes of Porites astreoides from Belize with lesions that do not resemble previously described coral diseases. The lines of evidence for the novelty of this syndrome include: the location of the lesions on the coral, the pattern of bleaching, the colonization of bare skeleton behind the disease front, and the characteristic thin, raised yellow band that has not previously been described. The bleaching associated with these lesions might suggest the presence of one of the many “white” coral diseases (for example, White Band type I or II, White Plague type I or II, Ulcerative White Spot, or Porites White Patch Syndrome). Of these, only White Plague type I has previously been reported in Caribbean Porites \n [43] . The location of lesions described here is in contrast to those of White Plague-like diseases, which are characterized by lesions with basal and peripheral locations [44] . In addition, the pattern of bleaching is not consistent with Porites White Patch Syndrome, which begins as bleaching only of the tissue between polyps [45] . The location and morphology of the lesions observed in Porites is more consistent with Yellow Band disease, however, the rate of disease progression of YBD is much slower, and it is a disease that is more typically seen in Montastraea corals [46] . Differences in the surface microbiomes of lesioned versus non-symptomatic corals were inconsistent. In general, Endozoicomonas dominated most of the bacterial communities, regardless of disease state, constituting as much as 93% of the total surface microbiomes in healthy corals. This is consistent with previous studies including those at the same geographic location and elsewhere in the Caribbean, where the dominance of Endozoicomonas in the microbiomes of asymptomatic specimens of Porites has been demonstrated through a variety of sequencing techniques, including 454 sequencing of the V3-V4 regions of 16S rRNA genes [31] , Illumina sequencing of the V6 [47] and V5 regions [32] , and Sanger sequencing of nearly full-length 16S rRNA genes [30] , [48] . Endozoicomonas is also a dominant member of the microbiomes of soft corals [49] , [50] , encrusting corals [51] , and photosynthetic sea slugs [52] . In contrast, characterization of the microbial community in background seawater has shown that dominant taxa in coral microbiomes are enriched from the rare biosphere of the surrounding seawater [47] . Therefore, it is reasonable to hypothesize that Endozoicomonas is an obligate member of the healthy surface microbiome of P. astreoides . The tightly controlled relative proportions of Endozoicomonas OTUs and the significant negative correlations between Endozoicomonas OTUs may also suggest that strains within the genus perform specific co-evolved roles. While most samples were dominated by Endozoicomonas , a cluster (Cluster 1) of mostly lesioned samples demonstrated a distinct shift in the microbial community composition, which included the almost complete loss of Endozoicomonas and its replacement with Gammaproteobacteria of the genera Stenotrophomonas and Pseudoxanthomonas , fungi belonging to the genus Alternaria , Bacteroidetes of the genus Weeksella , and Betaproteobacteria of the genera Tepidimonas and Achromobacter . The loss of Endozoicomonas and its replacement with a more diverse microbial community was also characteristic of disease outbreaks in a Mediterranean gorgonian [53] , and in general, previous studies comparing diseased versus healthy corals have demonstrated a decrease in Oceanospirillales and an increase of generalist taxa in diseased corals, though not the same generalists detected here [54] . In contrast, when Red Sea corals of the genus Acropora were experimentally challenged with the effects of eutrophication and overfishing, Endozoicomonas consistently accounted for two-thirds of the microbial populations and only more minor components of the microbiome responded to the treatments [55] . The loss of Endozoicomonas concurrent with the establishment of coral disease, but not with the stressors of nutrient loading and competition from macroalgae, emphasizes the potential role of this group in the maintenance of coral health. Although several studies have demonstrated clear differences in healthy versus diseased coral microbiomes, especially in White Plague Disease [25] , [56] – [60] , not all cases are clear-cut. For example, differentiation of healthy and diseased microbiomes in corals with Yellow Band Disease was evident in some studies [61] , but lacking in others [26] , [28] . This lack of consistency may, in part, reflect the large differences in the methodologies used. In some cases, researchers pooled sample types together [45] , [58] , which would mask the variability between individual samples of the same health condition. The microbial signature may also be dependent on what part of the coral microbiome was sampled (the surface mucus layer, polyp tissues, or coral skeleton), as some studies have shown more pronounced differences in the microbial composition of tissue slurries in healthy versus diseased corals compared to the surface mucus layer [57] , [62] , although none of these compartments of the coral microbiome can truly be sampled as discrete units [63] . Our sampling of the surface mucus layer by vigorous rubbing of the coral surface and aspiration with a syringe almost certainly includes polyp tissue and gastric contents. Even though the majority of microbiomes in Cluster 1 were those recovered from lesioned corals, Cluster 2 also included microbial communities recovered from lesioned corals. The lack of distinct clustering according to health status is consistent with the outcomes of studies of the Yellow Band Disease (YBD) on Montastraea faveolata in the Caribbean and Ctenactis crassa and Herpolitha limax in the Red Sea where environmental conditions and seasonal effects had a more pronounced effect on microbial communities than the presence of the YBD lesion [26] , [28] . Collectively, these observations support the hypothesis that coral diseases can be associated with members of the native microbiota that escape as-yet-unknown restrictions that may be related to nutrient availability or optimal growth conditions (such as temperature or pH) or to competitive and antagonistic interactions with other members of the microbiome or the coral host. Once freed of these restrictions, otherwise rare members of the microbiome may become opportunistic pathogens. Potentially opportunistic pathogens were detected in the highly altered microbiomes of Cluster 1, including the fungal genus Alternaria , members of which are known to be opportunistic plant pathogens [64] . Alternaria are common in multiple niches in Australian coral reefs, with no apparent preference for host substrate [65] , and have been associated with marine sponges [66] and soft corals [67] . Fungi have previously been identified as potentially opportunistic pathogens of corals stressed by environmental conditions, including other species of Porites \n [68] . Some species of Alternaria are known to produce secondary metabolites with antimicrobial properties [69] , [70] , which may play a fundamental role in the disruption of the native coral microbiota. While fungi in the phylum Ascomycota have previously been detected using metagenomic analysis of the healthy Porites astreoides microbiome, the dominant fungi detected belong to a different class (Sodariomycetes) than the Alternaria detected here [29] . While we cannot determine the abundance of Sodariomycetes in the Porites sampled here given the different sequencing methodologies used (i.e., shotgun metagenome sequencing versus amplified bacterial marker gene sequencing), the lack of Alternaria in the metagenomes of healthy P. astreoides corroborates our finding that Alternaria is absent or at low abundance in healthy Porites . \n Stenotrophomonas and Achromobacter were also common in the Cluster 1 samples, and members of both of these bacterial genera are known opportunistic pathogens involved in nosocomial infections of immunocompromised patients. The type species for the genus Stenotrophomonas , S. maltophilia , has been identified in biofilms from a wide range of habitats and has been implicated as either a primary or secondary pathogen in a variety of human diseases, particularly respiratory tract infections [71] . While not highly virulent, this species in particular is increasingly recognized as a concern in hospitals as it is a common contaminant of water supplies, hand soap, and medical equipment, resistant to biocides and heavy metals, and is emerging as a multiple-drug-resistant organism [72] . Stenotrophomonas was recently detected in both stony and soft corals from the Red Sea and was most abundant in corals at relatively disturbed sites that experienced higher light levels and higher water column nutrient concentrations [73] . Achromobacter has also been detected in a wide variety of soil and water samples as well as clinical infections, and comparative genomic analyses demonstrate that strains in this genus also have the potential for multidrug resistance and heavy metal resistance [74] . While phylogenetic proximity to known pathogens is not direct evidence that these groups are also coral pathogens, their abundance in the highly altered microbial community of lesioned Porites strongly suggests that they play a role in either creating the lesions or taking advantage of the damaged tissue to establish a secondary infection. In addition, the striking similarities in the characteristics of the genera Stenotrophomonas and Achromobacter suggest that biofilm formation and antimicrobial resistance play a role in the disruption of native commensal microbiota. Further tests involving the isolation of these groups from corals may yield further insights into their role in the establishment of disease in Porites astreoides . While Achromobacter was not detected in any samples from nonsymptomatic corals in September, both Stenotrophomonas and Alternaria were present at low levels (<0.2% of reads) in some, but not all of the nonsymptomatic microbiomes. Additional sequencing depth may have revealed that Achromobacter is also present as a rare member of the healthy coral microbiomes, thus we emphasize that the development of these unusual lesions in P. astreoides does not coincide with the appearance of specific taxa, but rather with the replacement of Endozoicomonas with generalist taxa that are potentially opportunistic pathogens. Significant positive correlations between the abundant groups in Cluster 1, including opportunistic pathogens, and Endozoicomonas imply that an antagonistic relationship does not exist. However, negative correlations were found between Endozoicomonas and members of the Vibrionaceae , which in turn were positively correlated with the opportunistic pathogens. Thus, the loss of Endozoicomonas may be a complex transition that results in higher abundances of Vibrionaceae and other opportunistic pathogens. It is reasonable to hypothesize that a stable microbial community associated with healthy corals is robust, and any destabilization of the microbiome, characterized here by the disappearance of Endozoicomonas , is associated with the eventual appearance of lesions. There is growing evidence that coral disease is the result of secondary infections of corals experiencing environmental stress. In the Caribbean, which is a hotspot for coral disease, the lack of spatial clustering in diseased corals suggests that these diseases do not follow a typical model for the spread of contagious diseases and are more likely to be opportunistic infections [22] . Previous work has shown that both the community composition and functional gene composition of the microbiomes of Porites astreoides shift when the coral is exposed to environmental stressors such as elevated temperatures, lowered pH, and increased nutrient levels [75] . Here, lesions in Porites were detected only in shallow waters and only during warm months, which is consistent with regional studies demonstrating a significant correlation between higher temperatures and higher prevalence of coral diseases [76] . Implications for the prevention and control of diseases in corals include the need to focus on primary environmental stressors such as elevated ocean temperatures, nutrients and ocean acidification."
} | 4,653 |
36275121 | null | s2 | 6,142 | {
"abstract": "No abstract available"
} | 5 |
38350871 | PMC10865589 | pmc | 6,145 | {
"abstract": "Alfalfa, an essential forage crop known for its high yield, nutritional value, and strong adaptability, has been widely cultivated worldwide. The yield and quality of alfalfa are frequently jeopardized due to environmental degradation. Lignin, a constituent of the cell wall, enhances plant resistance to abiotic stress, which often causes osmotic stress in plant cells. However, how lignin responds to osmotic stress in leaves remains unclear. This study explored the effects of osmotic stress on lignin accumulation and the contents of intermediate metabolites involved in lignin synthesis in alfalfa leaves. Osmotic stress caused an increase in lignin accumulation and the alteration of core enzyme activities and gene expression in the phenylpropanoid pathway. We identified five hub genes ( CSE , CCR , CADa , CADb , and POD ) and thirty edge genes (including WRKY s, MYBs , and UBPs ) by integrating transcriptome and metabolome analyses. In addition, ABA and ethylene signaling induced by osmotic stress regulated lignin biosynthesis in a contradictory way. These findings contribute to a new theoretical foundation for the breeding of high-quality and resistant alfalfa varieties. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10039-1.",
"conclusion": "Conclusion In summary, a combination of physiological, metabolomic, and transcriptomic analyses was performed to understand the lignin synthesis under osmotic stress in the leaves of alfalfa. Lignin content increased gradually under osmotic stress. The increase in enzyme activity promoted the accumulation of 5 metabolites. We identified 5 hub genes and constructed a co-expression network of lignin biosynthesis based on WGCNA analysis. In addition, ABA regulated lignin content positively, and ethylene regulated lignin content negatively under osmotic stress. These conclusions help us get a better understanding of lignin synthesis and regulation mechanism in alfalfa under osmotic stress, and provide new insight into the improvements of abiotic tolerance and quality of alfalfa. However, the function of the identified key genes needs to be further verified, and how ABA and ethylene regulate lignin needs to be further studied.",
"introduction": "Introduction As sessile organisms, plants have to adapt to changing environments. Osmotic stress can be caused by plants subjected to various abiotic stresses, including drought, salt, and low temperature stresses [ 1 ]. When plants are threatened by osmotic stress, the cells lose water seriously, which adversely affects the normal growth of plants. Abiotic-induced osmotic stress severely limits plant biomass production and poses significant threats to agricultural industries [ 2 ]. Consequently, it is necessary to investigate the response mechanism of plants under osmotic stress. Understanding of how plants respond to osmotic stress is important not only for basic biology but also for agriculture [ 1 ]. Some studies have shown that plants can resist adverse environmental stress by increasing lignin deposition [ 3 ]. Lignin is an important component of the plant cell wall, which is conducive to the growth, development, and water transport of plants, and it can also alleviate damage to plants by adverse external environments or pathogens [ 4 ]. MeRAV5 improved lignin accumulation to enhance drought stress resistance by promoting the activities of both MePOD and MeCAD15 in cassava [ 5 ]. Overexpression of MdSND1 increased the lignin content in apples and improved the salt and osmotic stress resistance [ 6 ]. Overexpressing of AgNAC1 enhanced the plants’ resistance to salt stress by increasing lignin accumulation in Arabidopsis [ 7 ]. Although lignin is beneficial to plant resistance to osmotic stress, knowledge of lignin response to osmotic stress is still limited. Previous studies based on the model plant Arabidopsis thaliana and the model woody plant poplar have identified 11 core enzymes related to lignin biosynthesis in plants [ 8 , 9 ]. Lignin synthesis was affected by enzyme activities and the expression levels of the genes that encode these enzymes. L-phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-hydroxycinnamate CoA ligase (4CL) are the three common enzymes that belong to the general phenylpropanoid pathway. The other eight enzymes, including caffeoyl shikimate esterase (CSE), hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), coumarate 3-hydroxylase (C3H), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT), caffeoyl CoA 3-O-methyltransferase (CCoAOMT), cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl CoA reductase (CCR), belong to lignin-specific pathway [ 10 ]. In addition, some transcriptional factors ( NAC and MYB ) are important to lignin synthesis [ 11 ]. Some of the NAC transcription factors were regarded as the switch of lignin synthesis by regulating the genes involved in cell wall biosynthesis in Arabidopsis [ 12 ]. Transcriptional activation of OsCCR10 by OsNAC5 enhanced the ability to resist drought stress by increasing lignin content in the root of rice [ 13 ]. Except for the NAC genes family, MYB transcription factors were extensively related to the transcriptional regulation of lignin. MYB58 and MYB63 positively regulated cell wall formation by lignin synthesis in Arabidopsis [ 14 ]. As a transcriptional activator of the lignin synthesis of populus, PtoMYB92 contributed to cell wall formation [ 15 ]. CmMYB15 regulated the biosynthesis of lignin in chrysanthemum [ 16 ]. CsMYB330 and CsMYB308 promoted fruit lignification by regulated expression of the Cs4CL1 gene in Citrus sinensis [ 17 ]. The synthesis and regulation of lignin have been studied in several plants, but how osmotic stress regulates lignin synthesis has not been systematically investigated. Alfalfa is an important leguminous forage with high nutritional value and strong adaptability, and it is an autotetraploid with a genome size of 2.738 Gb and 32 chromosomes, including eight homologous groups with four allelic chromosomes in each [ 18 ]. In China, alfalfa grows mostly in arid and semi-arid areas and often suffers from osmotic stress caused by drought or salt stress, which threatens the yield and quality of alfalfa. Although lignin can improve the ability of alfalfa to adapt to abiotic stress, it can also reduce the forage digestibility. Therefore, exploring the synthesis and regulation mechanism of lignin under osmotic stress is important. Previous studies have shown that an increase in l-phenylalanine content provides favorable conditions for lignin synthesis in alfalfa leaves, which is one of the main factors contributing to a decline in alfalfa RFV and quality [ 19 ]. However, how osmotic stress regulates lignin biosynthesis in leaves is unclear. The assimilates were synthesized in mesophyll cells through photosynthesis and then transported through the vascular systems to stems for growth [ 20 ] and structural carbohydrate (lignin and cellulose) biosynthesis [ 21 ]. The lignification of leaf vasculature affected water and nutrient transport and lignin accumulation in the stems. In this study, we explored the lignin synthesis mechanism in alfalfa leaves under osmotic stress by combining physiological, metabolomic, and transcriptomic analyses. We investigated the activities of key enzymes related to lignin biosynthesis as well as metabolite accumulation levels, identified key structural genes of lignin in response to osmotic stress, and constructed a gene co-expression regulatory network. We conducted combined metabolomic and transcriptomic analyses to identify genes involved in lignin biosynthesis under osmotic stress, providing a theoretical basis for the selection of target genes in the molecular breeding of alfalfa. Our findings provide new insights for breeding high-quality or improved-stress-tolerant alfalfa varieties.",
"discussion": "Discussion As an important component of the cell wall, lignin helps plants to resist adverse external environments, including biotic and abiotic stress. Drought, salt, and other abiotic stresses always cause osmotic stress, and the mechanism of how lignin responds to osmotic stress remains unclear. Here, the combination of physiological, transcriptional, and metabolic analysis was used to reveal the regulation mechanism of alfalfa lignin under mannitol-induced osmotic stress. In this study, after mannitol-induced osmotic stress for 1 d and 4 d, the lignin content of alfalfa leaves increased significantly. The increase in lignin accumulation might contribute to enhancing osmotic tolerance in alfalfa leaves. The lignin content decreased on 7 d with a higher level than CK. We speculated that mannitol treatment for 7 days may result in severe damage to plant physiological processes as the RWC of alfalfa is below 60%. Alternatively, the photosynthetic products or intermediate metabolites necessary for lignin synthesis were not sufficient for lignin synthesis under long-term osmotic stress. Several previous studies have found that drought or salt stress has a positive effect on lignin synthesis in different plants. The leaf lignin content of maize was increased significantly under severe and moderate drought stress, and lignin was used as an index for the evaluation of drought stress in maize [ 22 ]. Similarly, drought stress led to increased lignin accumulation in the primary root of chickpeas [ 23 ]. Our study not only suggested that osmotic stress can also regulated positively lignin biosynthesis in alfalfa, but also further elucidates the regulation mechanisms of lignin synthesis under osmotic stress at the level of metabolites and genes transcription expression. There are many intermediate metabolites involved in the lignin synthesis process. Previous research has pointed out that the content of these intermediate metabolites changes under abiotic stress [ 24 ]. Seven DAMs involved in lignin synthesis increased remarkably under salt stress in the root tissue of Sophora alopecuroides , including phenylalanine, cinnamic acid, ferulic acid, cinnamaldehyde, caffeylaldehyde, sinapyl alcohol, and coniferin [ 25 ]. Under low-temperature stress, the lignin content of tobacco leaves increased, and the accumulation of intermediate metabolites related to lignin synthesis such as p-coumaric acid, p-coumaroyl, and ferulic acid also increased [ 26 ]. In this study, we identified 5 DAMs that increased in alfalfa leaves, including cinnamic acid, p -Coumaric acid, ferulic acid, p -Coumaraldehyde, and sinapic acid. In our previous study, 8 DAMs related to lignin synthesis decreased under osmotic stress due to the dynamic changes in enzyme activities [ 27 ]. Comparatively, the increased metabolites in the leaves were also associated with changes in enzyme activities. PAL, C4H, and 4CL are three key enzymes of general metabolic pathways in phenylpropanoid biosynthesis; they catalyze phenylalanine to p-coumaroyl-CoA [ 28 ]. The increased accumulation of p -Coumaric acid and cinnamic acid was caused by a continuous increase in enzyme activity of PAL and C4H after 6 h osmotic stress. Similarly, the accumulation of ferulic acid, p -coumaraldehyde, and sinapic acid was due to the enzyme activity of COMT, CCR, and F5H increased. The decrease of 4CL enzyme activity which is induced by decreased expression level of 4CL ( MsG0580024209.01 ) reduces substrate (ferulic acid, p -coumaric acid, and sinapic acid) consumption, and it eventually led to an increase in the accumulation of these substrates. Similarly, the increase of p -coumaraldehyde was due to that CCR activity increased continuously and CAD activity decreased at 6 h. It was worth noting that all these enzyme activities increased after the short-term (6 h or 1 day) of osmotic stress and the metabolite contents increased after long-term (4 days or 7 days) osmotic stress. The reason might be that osmotic stress induced a quick improvement of the lignin synthesis-related enzyme activities, thus resulting in metabolite accumulation in alfalfa leaves. However, in our previous study, while the enzyme activities increased, the accumulation of DAMs decreased in the stems of alfalfa [ 27 ]. We speculated that phenylalanine, the original substrate for the phenylpropane pathway, was sufficient for lignin synthesis in the leaves, resulting in a great increase in cinnamic acid content. The redundant substrates were then transported to the stem for lignin accumulation. The decreased metabolites suggested that the lignin synthesis might be restricted in the stem due to the priority supply of substrates to the leaf. Lignin biosynthesis improved plant resistance to abiotic stress. Previous studies identified 28 DEGs belonging to eight families ( PALs , C4Hs , 4CLs , COMTs , CCRs , CADs , PODs , and UGTs ), which took part in lignin synthesis under salt stress in Sophora alopecuroides [ 25 ]. In our research, we found 59 DEGs involved in the lignin synthesis. They belonged to 12 gene families, four ( HCT , CCRs , CADs , UGTs ) of which were all upregulated, indicating that upregulation of these genes may be one of the most crucial factors in lignin synthesis when alfalfa encounters osmotic stress. Some previous studies have found that drought stress increased CCR protein abundance and lignin content in Leucaena seedlings stems, suggesting that CCR-catalyzed lignin synthesis may be critical for drought stress tolerance of Leucaena [ 29 ]. CmCAD2 was designated as an abiotic-stress-response gene that participates in lignin biosynthesis in oriental melons [ 30 ]. Two CsHCT genes were upregulated under the four stress treatments (cold, salt, drought, and MeJA stress) in tea plants [ 31 ]. In addition, all of the CCRs , CADs , and UGTs were also significantly upregulated in alfalfa stem tissue under osmotic stress [ 27 ], indicating that CCRs , CADs , and UGTs played an important role in both stems and leaves of alfalfa under osmotic stress. Meanwhile, CCR ( MsG0480018285.01 ), CADa ( MsG0180005813.01 ), and CADb ( MsG0280006638.01 ) were hub genes identified by WGCNA analysis. Peroxidase (POD) is an enzyme that has multiple functions, and it participated in several diffident plant physiological processes, involving stress resistance, oxidation, and polymerization of lignin monomers after they were transported to the cell walls [ 32 ]. Under cold stress, the POD gene expression was upregulated in the roots and leaves of sweetpotato [ 32 ]. Transcript abundance of POD in leaves was obviously increased by salt shock in Eutrema salsugineum [ 33 ]. In this research, CSE ( MsG0180006172.01 ) and POD ( MsG0380016745.01 ) were hub genes for lignin synthesis. In our previous study, these two genes were also hub genes related to lignin biosynthesis in alfalfa stems under osmotic stress [ 27 ]. WGCNA analysis identified 30 edge genes, which were supposed to be very important to the regulation of lignin synthesis under osmotic stress. The edge genes included MYB , WRKY , and genes encoded ubiquitin-specific protease. MYB15 was the homologous gene of AtMYB15 , which played an important role in lignin synthesis in effector-triggered immunity [ 34 ]. Among homologous genes of edge genes, AtMYB111 , AtMYB113 , and AtMYBD took part in the regulation of anthocyanin and flavonol in phenylpropane metabolism [ 35 , 36 ]. Notably, UBP7 , as the homologous genes of AtUBP7 , encoded a ubiquitin-specific protease associated with all five hub genes in leaves. We speculated that lignin biosynthesis was related to ubiquitination under osmotic stress. These results indicated that the lignin synthesis was regulated in multilevel under osmotic stress, including transcription and post-translational modification of proteins. GO analysis revealed that the “abscisic acid-activated signaling pathway” and “ethylene-activated signaling pathway” were enriched. To test the influence of ABA and ethylene on lignin biosynthesis, we measured the lignin accumulations under ABA/ACC treatment. Lignin content significantly increased by ABA or mannitol (MAN) treatment alone and the combination of MAN + ABA treatment. However, the lignin content was significantly reduced by ABA inhibitor (FLU). These results indicated that ABA signal molecules positively regulated lignin accumulation, and it was consistent with previous reports in Arabidopsis thaliana [ 37 ], apple [ 6 ], melon [ 38 ], and poplar [ 39 ]. However, another study suggested that the lignin accumulation in leaves was not affected by exogenous ABA in maize [ 40 ]. We suspected that the effect of ABA on lignin accumulation was specific in different species. Although there have been many studies on the relationship between lignin synthesis and ethylene, studies on lignin regulation by ethylene under abiotic stress remain unclear. In this research, under osmotic stress, the lignin biosynthesis of alfalfa leaves was inhibited by ACC, and it was relieved by AgNO 3 , an ethylene inhibitor. We speculate that osmotic stress may change the ethylene signaling and other metabolites, which have antagonistic or synergistic effects, to regulate the biosynthesis of lignin negatively. The influence of ABA and ethylene on lignin biosynthesis under osmotic stress is consistent with the results of our previous studies on stem tissue [ 27 ]."
} | 4,364 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.