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37712205 | PMC10578117 | pmc | 4,121 | {
"abstract": "Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C‐terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain‐swapped dimers formed by five‐helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four‐helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three‐state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non‐swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid‐induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.",
"introduction": "1 INTRODUCTION Silk fibers from orb‐web weaving spiders belong to the toughest materials in nature (Gosline et al., 1999 ; Heim et al., 2009 ; Vollrath and Knight, 2001 ). Web spiders use up to seven specialized glands to spin silk threads for various tasks including prey capture, reproduction, and shelter (Rising and Johansson, 2015 ; Vollrath and Knight, 2001 ). Spider silk is a protein‐based biopolymer that consists of fibroins, so‐called spidroins. Despite the differences in mechanical properties of the different silks they produce, the process of synthesis by the animal is conserved. At the beginning of the process, spidroins are stored as soluble proteins at high concentration in the ampulla of a gland where they form a viscous spinning dope (Heim et al., 2009 ; Vollrath and Knight, 2001 ). On demand, the dope passes through a narrowing, S‐shaped duct where spidroins experience a series of mechanical and chemical stimuli (Heim et al., 2009 ; Rising and Johansson, 2015 ; Vollrath and Knight, 2001 ). Shear forces align spidroins (Vollrath and Knight, 2001 ) and changes in salt composition and pH induce phase and structural transitions (Heim et al., 2009 ; Rising and Johansson, 2015 ). At the end of the duct, the solid silk fiber emerges from the exit spigot. Raman spectromicroscopy reveals gland‐specific differences in structural transitions of spidroins during their transformation into silk (Lefevre et al., 2011 ). Spidroins from major (Ma) and minor (Mi) ampullate glands, which form dragline and auxiliary silk, are natively unfolded under storage conditions. In the corresponding silks they are transformed into highly oriented β‐sheet or α/β‐structures. By contrast, spidroins from the flagelliform (Flag) gland, which forms capture spiral silk, undergo no structural transitions: they are highly disordered both under storage conditions and in solid silk (Lefevre et al., 2011 ). These remarkable differences in structural transformations are thought to originate from differences in sequence (Challis et al., 2006 ; Lefevre et al., 2011 ). The molecular details are only understood in parts. They are of interest for material scientists trying to infer mechanical properties from protein structural architectures, interactions, and energetics (Yarger et al., 2018 ). They are also of interest for biochemists trying to understand fundamental mechanisms of protein folding and protein structure–function relationships. The amino acid composition of spidroins is unusual compared to the one of conventional proteins (Vollrath and Knight 2001 ). The bulk of a spidroin consists of an intrinsically disordered central domain that contains repetitive peptide motifs of simple amino acid composition, typically enriched in Ala, Gly, Gln, Pro, and Ser (Heim et al., 2009 ; Vollrath and Knight, 2001 ). The unusual amino acid composition of the central domain extends into the globular folded N‐ and C‐terminal domains (NTD and CTD). NTD and CTD are five‐helix bundles that provide water‐solubility under storage conditions and connectivity in silk (Askarieh et al., 2010 ; Hagn et al., 2010 ). Sequences of the spidroin terminal domains are highly conserved indicating conserved and important functional roles (Eisoldt et al., 2012 ). Both NTD and CTD form all‐helical homo‐dimers, respectively, but their quaternary structures are very different (Askarieh et al., 2010 ; Hagn et al., 2010 ). They contain structural switches that are triggered by chemical stimuli within spinning ducts. One of these stimuli is a drop of solution pH from 7.6 in the ampulla to 5.7, and possibly below, at the end of the duct, which affects NTD and CTD differently (Andersson et al., 2014 ). The NTD is a monomer under storage conditions and forms a tight dimer upon solution acidification, a process that involves electrostatic interactions and conformational change (Askarieh et al., 2010 ; Gaines et al., 2010 ; Hagn et al., 2011 ; Heiby et al., 2019 ; Jaudzems et al., 2012 ; Kronqvist et al., 2014 ; Ries et al., 2014 ; Schwarze et al., 2013 ). NTD dimerization is thought to polymerize spidroins. The CTD, on the other hand, is a dimer already under storage conditions connecting two spidroins permanantly (Hagn et al., 2010 ). During passage of spidroins through an acidified zone within the spinning duct the CTD gets destabilized (Andersson et al., 2014 ). Subsequent partial unfolding of the CTD induces formation of β‐sheets, which is the dominant secondary structure in spider silk. The process is associated with phase transition (Andersson et al., 2014 ; Bauer and Scheibel, 2017 ; Gauthier et al., 2014 ; Hagn et al., 2010 ). The sequences of CTDs are less conserved compared to the ones of NTDs (Eisoldt et al., 2012 ). However, a pH sensitive module is conserved across the entire spider silk gene family (Strickland et al., 2018 ). Structures of CTDs from Ma, Mi, and aciniform glands are highly similar and exhibit a characteristic domain swap where the C‐terminal helices within the dimer exchange between monomer subunits thus forming a macromolecular clamp (Andersson et al., 2014 ; Gao et al., 2013 ; Hagn et al., 2010 ; Wang et al., 2014 ). Within the family of CTDs, sequences from the Flag gland differ (Challis et al., 2006 ). A recently determined structure of a Flag spidroin CTD lacks domain swap but its secondary and tertiary structure is otherwise similar (Li et al., 2022 ). Domain swap is a phenomenon in protein dimerization where monomers exchange parts of their structure upon association. It was first discovered by Eisenberg and colleagues and suggested as an evolutionary mechanism in protein oligomerization (Anonymous, 1994 ; Bennett et al., 1994 ). Domain swap increases the area of a dimerization interface considerably and is thus suggested to exert a marked stabilizing effect on protein assemblies (Mackinnon et al., 2013 ; Newcomer, 2002 ). It is further thought to regulate protein function and aggregation (Newcomer, 2002 ; Rousseau et al., 2012 ), and plays an important role in β‐amyloid fibril formation (Nelson and Eisenberg, 2006 ). The folding mechanism of domain swap and how it serves to regulate function are elusive (Gronenborn, 2009 ). Folding of spidroin CTDs from various glands and species has been studied and is reported to proceed via a two‐state transition, but with indications for or the observation of residual structure in denatured states (Andersson et al., 2014 ; Bauer and Scheibel, 2017 ; Gao et al., 2013 ; Gauthier et al., 2014 ; Hagn et al., 2010 ). In these studies, urea was applied as denaturant in chemical denaturation experiments. The application of urea led to apparently incomplete unfolding, hampering mechanistic, quantitative studies. An exception was a spidroin CTD from the Ma gland of the nursery web spider Euprosthenops australis that fully unfolded in urea and was found to self‐assemble via a three‐state mechanism (Rat et al., 2018 ). Here, we investigated the folding and dimerization of a family of five homologous spidroin CTDs originating from various species or glands in a comparative manner. We applied the stronger chaotrope guanidinium chloride (GdmCl) instead of urea. We observed full unfolding of all CTDs, which allowed us to explore their entire free energy surfaces. We found a three‐state mechanism of folding and dimerization via a dimeric folding intermediate that was conserved across homologues. The conformational stabilities of native and intermediate states varied between homologues. The CTD from the Flag gland that lacked domain swap was most stable and showed fast kinetics of folding. The observed differences can be explained by the entropic penalty associated with domain swap with functional consequences.",
"discussion": "3 DISCUSSION The globular C‐terminus of spidroins is a highly conserved (Strickland et al., 2018 ), pH‐sensitive connectivity module. CTDs have an established role in inducing conformational changes during silk formation facilitated by acid‐induced CTD destabilization, leading to formation of β‐sheet secondary structures (Andersson et al., 2014 ; Bauer and Scheibel, 2017 ; Eisoldt et al., 2012 ; Gauthier et al., 2014 ; Hagn et al., 2010 ). Folding studies can shed light on the underlying mechanisms. Previously, urea was applied as a chaotrope in chemical denaturation experiments of CTDs, which led to incomplete unfolding and the observation of residual structure in denatured states (Andersson et al., 2014 ; Bauer and Scheibel, 2017 ; Gao et al., 2013 ; Gauthier et al., 2014 ; Hagn et al., 2010 ; Li et al., 2022 ). In agreement with these results, we found that four of the five investigated CTDs did not fully unfold in solutions containing concentrations of up to 10 M urea (Figure 3 ). The ability of CTDs to resist such high concentrations of denaturant underscores their remarkable strengths as dimers serving as connectivity modules in silk. Our results show that full unfolding can be achieved by applying the stronger denaturant GdmCl, which allowed us to explore their entire free energy surfaces of folding and dimerization (Figure 2 ). We found a three‐state mechanism of folding and dimerization via a partially structured, dimeric intermediate that was conserved across five homologues. Intermediates are often transient species in the folding of small protein domains and difficult to detect (Brockwell and Radford, 2007 ). But here, the intermediates were exclusively populated in denaturation experiments where they were evident as well‐defined intermediate‐state baselines in denaturation curves. Under oxidizing solution conditions, as they exist in web spiders' spinning ducts, partially folded intermediates of CTDs from the Ma glands were the end points of unfolding (Figure 2a–c ). Previously, equilibrium and kinetic experiments carried out on the Ea‐MaSp1‐CTD indicated that the first step in folding was the rapid formation of a tight dimer core followed by slow folding of peripheral helices in the second step (Rat et al., 2018 ). Given that the core of CTD dimers consists of two long, often covalently linked helical stalks, it is reasonable to assume that peripheral helices unfold first during acid‐induced denaturation of the native dimer in a spinning duct. The observed 50%–60% loss of helix in denatured or intermediate states compared to the native state, observed here, adds up with relative sum of residues of the peripheral helices H1–H3 or H1 and H2, and H5, seen in structures (Andersson et al., 2014 ; Gao et al., 2013 ; Hagn et al., 2010 ; Wang et al., 2014 ). Our mutagenesis experiments indicate that H1 and H2 of native Lh‐MaSp1‐CTD unfold during formation of the intermediate (Figure 5 ). The fact that constructs containing mutations in the C‐terminal helix H5 failed to express indicated the importance of this C‐terminal helix for structural integrity of the dimer. H5 swaps domains in most of the spidroin CTD dimers characterized so far. Proteins that swap domains upon dimerization contain a pre‐evolved association interface that is shared between monomers (Anonymous, 1994 ; Bennett et al., 1994 ). In spidroin CTDs the swap of C‐terminal H5 creates an interface for docking of N‐terminal helices H1 and H2 on the other monomer (Figure 6 ). H5, H1, and H2 are likely to unfold cooperatively because the helices stabilize each other through quaternary interactions. Transition N 2 → I 2 of domain‐swapped spidroin CTDs thus likely involves unfolding of H1, H2, and H5. FIGURE 6 Domain swap of C‐terminal helices creates an interface for docking of N‐terminal helices. Structure of the Lh‐MaSp1‐CTD with monomer subunits colored blue and cyan. Visible helices H1–H4 of one subunit and swapped H5 from the other are indicated. The swapped C‐terminal H5 from the other subunit creates a scaffold for folding and docking of N‐terminal H1 and H2. Structural studies show that domain swap of protein dimers increases the buried surface area of the dimerization interface by ~72% on average (MacKinnon and Wodak, 2015 ). This substantial increase of buried surface area is thought to increase the stability of the assembly and suggests a biological role of domain swap in protein stabilization (Mackinnon et al., 2013 ; MacKinnon and Wodak, 2015 ). We calculated the areas of dimerization interfaces of our investigated CTDs from structures using the PISA tool of the Protein Data Bank in Europe (PDBePISA, EMBL‐EBI) (Figure 7 ). The domain‐swapped dimerization interfaces of CTDs from the Ma glands were 70%–75% higher than the one of the Av‐Flag‐CTD, in agreement with the reported ~72% expectation value (MacKinnon and Wodak, 2015 ). But the free energies of unfolding and dissociation (Δ G \n N2‐2D ) of the five homologues were within error (Figure 7 ). The stability of the Av‐Flag‐CTD that lacks domain swap was highest among the five homologues, with reference to the formation of the intermediate (Δ G \n N2‐I2 ). This is contrary to the expectation of a stabilizing effect of domain swap on protein stability (Mackinnon et al., 2013 ; MacKinnon and Wodak, 2015 ). The finding can be explained by the concept of contact order in protein folding, which suggests that the rate constant of folding is largely determined by the topology of the native structure (Grantcharova et al., 2001 ). Contact order is defined as the average distance in sequence between residues that form native contacts (Plaxco et al., 1998 ). The entropic cost of folding increases with increasing sequence separation of interacting residues. Protein structures that exhibit high contact order fold slowly because of this entropic penalty (Grantcharova et al., 2001 ). In domain swap, native contacts are created between residues of two independent polypeptide chains. This can be viewed as an extreme case of high contact order because a sequence separation is not defined. Our kinetic experiments showed that the rate constant of folding of the Av‐Flag‐CTD was k \n f = 6400 ± 1900 s −1 , which is four orders of magnitude higher than the value determined for a Ma spidroin CTD of k \n f = 0.78 ± 0.07 s −1 (Rat et al., 2018 ). The rate constant of unfolding of the Av‐Flag‐CTD was k \n u = 0.7 ± 0.1 × 10 −3 s −1 , which compares to k \n u = 9 ± 2 × 10 −3 s −1 measured for a Ma CTD (Rat et al., 2018 ). The higher stability of the Av‐Flag‐CTD compared to the Ma homologue thus originates largely from its higher rate constant of folding (Δ G = − RT ln( k \n f / k \n u )). The discrepancy in folding rates is explained by the loss of conformational entropy associated with domain swap. An alternative explanation is that steric hindrance, arising from the volume excluded by the partially folded dimer core formed by helices 3 and 4 in I 2 , slows folding and docking of C‐terminal helix 5 onto the other subunit. FIGURE 7 Dimerization interface areas and stabilities of spidroin CTDs. Areas of the dimerization interfaces, calculated using the PISA tool of the Protein Data Bank in Europe (PDBePISA, EMBL‐EBI), are shown in red. The changes of free energy of the transitions N 2 to I 2 (Δ G \n N2‐I2 ) and N 2 to 2D (Δ G \n N2‐2D ), determined from chemical equilibrium denaturation data recorded in oxidizing and reducing solutions at pH 7.0, are shown in blue and cyan, respectively. Error bars are propagated standard errors from regression analysis. Equilibrium m ‐values derived from chemical denaturation experiments contain structural information. In chemical denaturation of single‐domain proteins, the equilibrium m ‐value correlates with the amount of surface exposed to the solvent upon unfolding (Myers et al., 1995 ). In chemical denaturation of protein dimers, however, the m ‐value is more difficult to interpret because unfolding involves protein dissociation. Among the five CTDs studied, the Av‐Flag‐CTD was the only homologue that showed a mono‐molecular three‐state transition with well separated native, intermediate, and denatured states populated by a covalent dimer (Figure 2e ). Here we could interpret the m ‐values with confidence. In all other homologues, the covalent dimers resisted unfolding to fully denatured dimers and showed apparent two‐state transitions to structured denatured or intermediate states, except of the Lh‐MiSp1‐CTD that contained no Cys and dissociated to unfolded monomers (Figure 2a–d ). For the mono‐molecular three‐state unfolding transition of the Av‐Flag‐CTD, the m ‐value of the first transition was substantially higher than the m ‐value of the second transition (Table S1 ). This indicated that large parts of buried surface area became exposed to solvent in the first unfolding transition. Strikingly, this difference in m ‐values between the first and second transitions was also evident in three‐state unfolding of all homologues studied under reducing conditions (Table S2 ). We found that the m ‐values of the second step were essentially independent on protein concentration (the s.d. of measurements at five different protein concentrations was ±0.1 kcal M −1 mol −1 ). The higher s.d. of the mean m ‐value of the Av‐Flag‐CTD of ±0.3 kcal M −1 mol −1 can be explained by the fact that the first and second transitions appeared to merge, which led to higher errors of thermodynamic parameters extracted from fits to the data (Table S2 , Figure 2e ). Our data of five homologues indicated that the largest part of buried surface area of native CTD dimers becomes exposed to solvent in the first unfolding transition. This unfolding transition is relevant for acid‐induced destabilization and subsequent partial unfolding of the CTD along a spider's spinning duct, which is thought to induce structural transitions in spidroins (Andersson et al., 2014 ; Bauer and Scheibel, 2017 ; Eisoldt et al., 2012 ; Gauthier et al., 2014 ; Hagn et al., 2010 ). The high m ‐value associated with transition N 2 to I 2 indicates that large parts of structure of CTD dimers are unfolded in I 2 and ready to refold into, for example, β‐sheet secondary structure. In conclusion, we found a three‐state mechanism of folding and dimerization that was conserved across homologous spidroin CTDs. In the dimeric intermediate state, large parts of peripheral helices are unfolded. The entropic penalty of domain swap of CTD dimers from Ma and Mi glands, serves to destabilize the assembly for functional purpose. This destabilizing effect contrasts with the common view of a stabilizing effect domain swap exerts on protein dimers through domain swap. Our finding of a destabilizing effect of domain swap of spidroin CTDs is supported by results from structural studies. Soluble spidroins from Ma and Mi glands that contain domain‐swapped CTDs undergo conformational transitions into β‐sheet secondary structures found in silk fibers (Lefevre et al., 2011 ). The CTD from the Flag gland, instead, lacks domain swap, and the corresponding spidroin undergoes no structural transitions during silk formation (Lefevre et al., 2011 ). A conformationally more stable CTD lacking domain swap impedes structural transitions."
} | 5,228 |
22144155 | null | s2 | 4,122 | {
"abstract": "The BioCyc database collection at BioCyc.org integrates genome and cellular network information for more than 1,100 organisms. This method chapter describes Web-based tools for browsing metabolic and regulatory networks within BioCyc. These tools allow visualization of complete metabolic and regulatory networks, and allow the user to zoom-in on regions of the network of interest. The user can find objects of interest such as genes and metabolites within the networks, and can selectively examine the connectivity of the network. The EcoCyc database within the BioCyc collection has been extensively curated. The descriptions within EcoCyc of the Escherichia coli metabolic network and regulatory network were derived from thousands of publications. Other BioCyc databases received moderate levels of curation, or no curation at all. Those databases receiving no curation contain metabolic networks that were computationally inferred from the annotated genome sequences of each organism."
} | 247 |
30970685 | PMC6432116 | pmc | 4,124 | {
"abstract": "A significant amount of academic and industrial research efforts are devoted to the encapsulation of active substances within micro- or nanocarriers. The ultimate goal of core–shell systems is the protection of the encapsulated substance from the environment, and its controlled and targeted release. This can be accomplished by employing “stimuli-responsive” materials as constituents of the capsule shell. Among a wide range of factors that induce the release of the core material, we focus herein on the light stimulus. In polymers, this feature can be achieved introducing a photo-sensitive segment, whose activation leads to either rupture or modification of the diffusive properties of the capsule shell, allowing the delivery of the encapsulated material. Micro- and nano-encapsulation techniques are constantly spreading towards wider application fields, and many different active molecules have been encapsulated, such as additives for food-packaging, pesticides, dyes, pharmaceutics, fragrances and flavors or cosmetics. Herein, a review on the latest and most challenging polymer-based micro- and nano-sized hollow carriers exhibiting a light-responsive release behavior is presented. A special focus is put on systems activated by wavelengths less harmful for living organisms (mainly in the ultraviolet, visible and infrared range), as well as on different preparation techniques, namely liposomes, self-assembly, layer-by-layer, and interfacial polymerization.",
"conclusion": "6. Conclusions Significant progress in the design and the synthesis of light-responsive polymer micro- and nanocapsules has been made in recent years. Diversification of capsule preparation techniques and fine-tuning of materials chemical design provide an almost infinite number of strategies to obtain a customer-tailored application. However, many challenges need to be addressed, concerning both academic research and industrial application. Understanding the principles of the mechanisms at the basis of these stimuli-responsive materials is essential for developing novel encapsulation, release, and targeting methods. The ultimate challenge for light-triggered delivery of drugs or other active agents in biological environments is to grant the use of biocompatible materials and un-harmful release process in use. Among the wide variety of photosensitive capsules available, a sensitive factor is the choice of an appropriate size range of delivery systems. Microcapsules, for example, have been widely studied and exploited in commercial applications for their facile preparation and characterization. On the other hand, biological application, such as circulation or cellular uptake experiments, have desperate need of nanocapsules. Research and development in nano-sized range is currently experiencing a burst development and is in constant need for new carriers to further impact theranostics, nanomedicine and drug delivery.",
"introduction": "1. Introduction In recent years, a growing interest has been focused on micro- and nano encapsulation due to their fruitful applications in controlled release of drugs [ 1 ], active agents [ 2 ], catalysts [ 3 ], and paints [ 4 ], as well as in synthetic nano-reactors engineering [ 5 ]. Academic and industrial research is particularly interested in so-called “environmentally responsive” materials, able to respond to an external stimulus (e.g., temperature, pH, light, electric or magnetic field) by modifying one or more of their intrinsic properties. For their adaptive features, these materials are often called smart [ 6 , 7 ]. One of the most challenging aspects of micro- and nano-encapsulation is the obtainment of a controlled and modulated release of the encapsulated—or core—material that can be achieved using smart materials as components of the capsule shell [ 8 ]. The design and development of high-sensitive systems, able to smartly recognize an external triggering factor and to respond by modifying their own structure, is the ultimate purpose of scientists all over the world. For this purpose, polymeric materials are particularly suitable for technical applications because they are versatile and their properties can be easily tailored depending on the final use. Many external stimuli, such as pH [ 9 ], temperature [ 10 , 11 ], biological molecules [ 12 ], and redox reactions [ 13 ] have been employed to effect capsule permeability or induce capsule disruption, enabling the release of the encapsulated material. Light (infrared, UV radiation or simply sunlight) is certainly the most compelling external stimulus, because it can be delivered without direct contact, thus representing one of the few remote-control triggering factors available [ 14 ]. Like many promising technologies, photo-responsive systems have been inspired by nature, which has evolved many complex biological systems able to exploit light as an external source of energy and information. For example, the light-induced cis–trans isomerization of the retinal molecule triggers a number of events, including a change in the conformation of the opsin protein to which is bound, leading to a neural signal and ultimately to the perception of light [ 15 ]. Mimicking natural structures, photo-responsive polymers can be obtained introducing photo-sensitive moieties in the polymeric backbone or in the side chains. Among the best performing photo-sensitive molecules, azobenzene [ 16 ], stilbene [ 17 ], and spiropyrans [ 18 ] stand out. The photoactivity of each of these functional groups is based on the existence of two interconvertible isomers. Upon light irradiation, typically in the ultraviolet range, the molecules undergo a conformational rearrangement. In the case of azobenzene and stilbene, this alteration is expressed by variations in the molecular symmetry from a thermally stable trans ( E ) orientation to a less favorable cis ( Z ) orientation ( Figure 1 a,b) [ 19 ]. In spiropyrans, the irradiation induces a ring-opening reaction that leads to the formation of the isomeric merocyanine form, as shown in Figure 1 c [ 18 ]. One of the most interesting features of such photochromic materials is that isomerization is usually accompanied by molecular changes in physical properties such as polarity, viscosity and absorbance as well as macroscopic changes in material properties such as thickness, wettability and stability [ 20 ]. The presence of photo-responsive moieties in the capsule shell can therefore affect permeability of capsules or even lead to their disruption [ 21 ]. A key factor to take into account when designing photo-responsive micro- and nanocapsule systems is the wavelength of the light used to trigger the release. For outdoor use or other applications in which direct contact between light and capsules is granted, it is theoretically possible to employ any wavelength required by the photochromic materials that constitute the capsules shell. However, with regard to biomedical applications, the skin penetration depth of the light source involved in the release is the factor that determines the appropriate use of the capsules. The optical behavior of human skin upon light irradiation has been vastly studied and reviewed [ 22 ]. UV and visible light are reported having short penetration (few micrometers) depth and are most suitable for topical uses; on the contrary, near infrared light has a higher skin penetration depth of few millimeters and it could therefore be employed in internal delivery applications. This review intends to give an overview on recent advances in the preparation of light-responsive polymeric capsules. Different preparation technologies will be discussed in detail, including interfacial methods (interfacial polymerization and phase inversion precipitation), template methods, and self-assembly methods. Capsules properties such as size, morphology and release behavior will also be described, with a view on the envisaged target applications."
} | 1,984 |
35104147 | PMC8851890 | pmc | 4,131 | {
"abstract": "Superhydrophobic\nsurfaces have attracted considerable attention\nbecause of their unique water-repellency and their wide range of applications.\nThe conventional method to characterize the surface wetting properties\nof surfaces, including superhydrophobic surfaces, relies on measuring\nstatic and dynamic contact angles, and sliding angles of water drops.\nHowever, because of the inhomogeneities inherently present on surfaces\n(smooth and textured), such optical methods can result in relatively\nlarge variability in sliding angle measurements. In this work, by\nusing a force-based technique with ±1 μN sensitivity, the\nfriction force between water drops and various surfaces is measured.\nThe friction force can then be used to accurately predict the sliding\nangle of water drops of various sizes with improved consistency. We\nalso show that the measured friction force can be used to determine\nthe critical drop size below which a water drop is not expected to\nslide even at a tilt angle of 90°. The proposed technique to\ncharacterize the wetting properties of surfaces has a higher accuracy\n(between 15% and 65%, depending on the surface) compared to optical\nmethods.",
"conclusion": "4 Conclusions Although conventional optical-based water\nsliding angle measurements\nare quick and easy to perform and are commonly used to characterize\nthe wetting properties of surfaces, standard deviations in measurements\ncan be quite significant. The suggested force-based technique can\npredict SAs with less variability by probing a larger area of a surface\nthereby minimizing the influence of localized surface imperfections.\nDifferent drop sizes (3–50 μL) and five different surfaces\nwere explored to test the limitations of the force-based technique.\nIt was found that the technique was suitable for surface wettability\ncharacterization of all surfaces. Deviations between measured and\npredicted sliding angles at smaller drop sizes were a result of the\nring drop probe distorting the drop profile. We conclude that force-based\ndynamic friction measurements between water drops and surfaces ought\nto be used to more accurately characterize surface wetting properties.",
"introduction": "1 Introduction Lotus leaf surfaces have inspired scientists to gain a better understanding\nof the basic science and mechanisms behind their water-repellant properties\nand to fabricate biomimetic superhydrophobic (SH) surfaces. 1 Although the fabrication of SH surfaces can be\ntraced back to 1907 by Ollivier, 2 the concept\nof superhydrophobicity did not gain significant attention until this\nphenomenon was described in Lotus leaves. 3 The superhydrophobic property originates from the micro- and nanoscale\nstructures as well as the low surface energy waxy coating. 1 Various potential industrial applications have\nbeen proposed for SH surfaces. For example, superantiwetting textile\nsurfaces can be designed with self-cleaning, 4 self-healing, 5 antibacterial, 6 oil/water separation, 7 UV-blocking, 8 flame-retardant, 9 and photocatalytic 10 properties. Static water contact angle (WCA) and sliding (roll-off)\nangle (SA) have been proposed as two conventional wetting parameters\nto characterize the water repellency of surfaces. 1 , 11 The conventional method of measuring WCA and SA, that is, using\na goniometer, 12 − 21 is relatively fast and easy. However, optical-based methods have\nbeen shown to be prone to errors in WCA measurement. For example,\nin the case of a SH surface (i.e., WCA > 150°), the misplacement\nof the baseline boundary between the three phases can result in more\nthan a 10° error in WCA measurements. 22 SA measurements are also susceptible to errors associated with surface\ninhomogeneities originating from surface defects and contamination.\nIn a typical SA measurement, a water drop (∼10–20 μL)\nis placed on a surface residing on a tilt stage. The tilt angle of\nthe stage is then increased until the water drop begins to slide.\nHowever, any surface defect or contamination locally present at the\nwater drop/surface interface can pin the 3-phase contact line thus\nresulting in larger SAs. As a result, SA measurements can have relatively\nlarge standard deviations thereby preventing reliable differentiation\nbetween the wetting properties of surfaces. Several research\ngroups have explored force-based techniques to\ncharacterize the wetting properties of surfaces. 22 − 25 In a force-based measurement,\na water drop is sheared against a surface over a predetermined distance\nand velocity, while the friction force is monitored. Since the friction\nforce over the entire sheared distance is recorded, considering the\naverage friction force minimizes contributions from local inhomogeneities\nthereby providing a more accurate surface wetting characterization.\nSeveral instruments have been developed or adapted to measure the\nadhesion, friction, snap-in and pull-off forces of water drops on\nsurfaces thereby allowing for the characterization of surface wetting\nproperties. 26 − 29 R. Tadmor et al., 30 for the first time,\nmeasured the lateral adhesion force between a drop and a surface,\nusing a centrifugal adhesion balance. They successfully measured the\nlateral adhesion (or static friction) force as a function of normal\nforce acting on a water drop and the rest time of the drop before\nsliding. Yao et al. 31 used a similar setup\nand proposed a force-based model to calculate the SA from force measurements\nwith a ±1 μN sensitivity. Conventional SA measurements\ncannot be used to characterize the wettability of surfaces when the\ndrop does not slide even at a 90° tilt, either because of pinning\nevents on the surface or the drop weight being insufficient to overcome\nthe threshold force. K. Shi et al. 32 proposed\na new technique to accurately characterize the wetting properties\nof surfaces. They utilized a capillary sensor (with ±0.7–2\nμN sensitivity) attached to a water drop to measure the friction\nforce between the drop and solid surfaces. By recording the capillary\ndeflection, the friction force was extracted based on Hooke’s\nlaw. In this paper, a force-based approach is proposed to predict\nthe\nSA of water drops on surfaces with greater accuracy compared to optical-based\ntechniques. Compared to the use of a capillary sensor, the proposed\ntechnique can measure a higher magnitude of friction forces because\nof the greater capillary interaction between a water drop and ring\nprobe. The approach relies on measuring dynamic friction forces of\nwater drops sliding on surfaces as shown in Figure 1 . The fact that the measurement is performed\nover a relatively large shear distance thereby probing the wetting\nproperties of the surface over a large area allows for greater accuracy\ncompared to conventional optical-based measurements of the SA. Figure 1 Side-view optical\nimage of a 20 μL water drop (A) sliding\non a hydrophobic surface at a tilt angle α. When the drop begins\nto slide, the SA is equal to α. (B) sheared on a hydrophobic\nsurface at a velocity V and corresponding applied force F x . F ∥ is the friction force at the interface of the water drop and the\nsurface.",
"discussion": "3 Results and Discussion The force component\non a stationary drop acting parallel to a tilted\nsurface is mgsin(α) (see Figure 1 A) where m is the mass of the drop and g is the gravitational\nacceleration. As the tilt increases, this force increases until it\nexceeds the static friction force at which point the drop begins to\nslide. Figure 3 shows\na plot of the force component acting parallel to a tilted surface.\nSuperimposed in the plot are the measured friction forces (dotted\nlines) between similar sized drops and an OTS-modified silicon surface.\nFor a given drop size, the intersection of the friction force measured\nand the solid curve (i.e., F ∥ = mgsin (α)) provides the predicted sliding angle (PSA).\nThe PSAs for a 50 μL and a 20 μL water drop on an OTS-modified\nsilicon surface are 8.6° and 15.1°, respectively. As expected,\nsmaller drops require larger tilt angles to initiate sliding. However,\nwhen the drop is too small (for e.g., 3 μL), the force component\nrequired to initiate movement is never achieved. In other words, the\nfriction force (approximately 33 μN, see red dotted line) is\ngreater than the force component provided by the drop’s own\nweight. An additional force (>3 μN) would be required to\ncause\nthe 3 μL drop to slide on the 90° tilted OTS-modified silicon\nsurface. Figure 3 Plot of force versus angle for three drop sizes. The solid lines\ncorrespond to the force component acting parallel to the surface (i.e.,\nmgsin(α)) originating from the weight of the drop. The dotted\nlines correspond to the friction force measured while a drop is sheared\nagainst an OTS-modified Si surface. Figure 4 shows a\ncomparison of the measured sliding angles (MSAs) and predicted sliding\nangles (PSAs) of a 20 μL drop on various surfaces. The error\nbars correspond to the standard deviation from at least five measurements.\nBased on the magnitude of the error bars, the force-based technique\nhas less variability in predicting the SA on the hydrophobic surfaces\nincluding OTS-modified Si wafer, OTS-modified glass and PTFE. In the\ncase of the PTFE surface, the force-based technique reduced the standard\ndeviation of the SA from ±5.51° to ±2.87°. The\nadvantage of the force-based technique over the conventional technique\nis further demonstrated when comparing SH surfaces (i.e., SH_20_30\nand SH_20_20). Using conventional methods for SA measurements of SH\nsurfaces, an overlap was observed between the results; the minimum\nMSA for the SH_20_20 surface was approximately equal to the maximum\nMSA for the SH_20_30 surface (see SI Table S1 ). However, by using the force-based technique, one can differentiate\nthe values of the SAs between the SH surfaces. On average, the standard\ndeviation of SA measurements was reduced by ∼42% for hydrophobic\nand ∼58% for SH surfaces. Figure 4 Plot of the measured and predicted sliding\nangles of a 20 μL\ndrop on various surfaces. SH_20_30 and SH_20_20 are superhydrophobic\ntextured surfaces, whereas the other surfaces are flat hydrophobic\nsurfaces. To test the limitations of the\nforce-based technique, the latter\nwas used to predict the SA for water drops of various sizes ( Figure 5 ). A smooth hydrophobic\nOTS-modified Si wafer was selected as the model surface. Based on\nthe data, a good agreement was found between the PSA and the MSA for\ndrop sizes larger than 6 μL. However, the difference between\nthe MSA and PSA increased for smaller drop sizes (i.e., 4 μL\nand 5 μL). A possible explanation for this difference is that\nthe ring drop holder has a larger influence on the shape of smaller\ndrops. Small drops are distorted from their spherical shape thereby\naffecting the contact area between the water drop and the surface\nin the measurements. We again note the improved accuracy of the force-based\nmeasurement compared to the conventional technique as shown by the\nsmaller standard deviations obtained for the PSA measurements. Figure 5 Plot showing\nthe comparison between predicted and measured sliding\nangles for various water drop sizes on an OTS-modified silicon wafer. Another factor to consider is the fact that certain\nsamples have\nsignificant threshold (or static) friction forces (i.e., the maximum\nfriction force attained before sliding occurs) that are larger than\nthe dynamic friction force. This was not the case with the samples\nused in this study, but we propose that for samples which show a distinct\nthreshold friction force, the latter ought to be used (instead of\nthe dynamic friction) to calculate the predicted sliding angle. A\nseries of start–stop experiments on a single force-based measurement\ncan provide multiple data points for the threshold friction force."
} | 2,932 |
33963405 | PMC8290118 | pmc | 4,132 | {
"abstract": "Abstract Modern accounts of eukaryogenesis entail an endosymbiotic encounter between an archaeal host and a proteobacterial endosymbiont, with subsequent evolution giving rise to a unicell possessing a single nucleus and mitochondria. The mononucleate state of the last eukaryotic common ancestor (LECA) is seldom, if ever, questioned, even though cells harboring multiple (syncytia, coenocytes, and polykaryons) are surprisingly common across eukaryotic supergroups. Here, we present a survey of multinucleated forms. Ancestral character state reconstruction for representatives of 106 eukaryotic taxa using 16 different possible roots and supergroup sister relationships, indicate that LECA, in addition to being mitochondriate, sexual, and meiotic, was multinucleate. LECA exhibited closed mitosis, which is the rule for modern syncytial forms, shedding light on the mechanics of its chromosome segregation. A simple mathematical model shows that within LECA’s multinucleate cytosol, relationships among mitochondria and nuclei were neither one-to-one, nor one-to-many, but many-to-many, placing mitonuclear interactions and cytonuclear compatibility at the evolutionary base of eukaryotic cell origin. Within a syncytium, individual nuclei and individual mitochondria function as the initial lower-level evolutionary units of selection, as opposed to individual cells, during eukaryogenesis. Nuclei within a syncytium rescue each other’s lethal mutations, thereby postponing selection for viable nuclei and cytonuclear compatibility to the generation of spores, buffering transitional bottlenecks at eukaryogenesis. The prokaryote-to-eukaryote transition is traditionally thought to have left no intermediates, yet if eukaryogenesis proceeded via a syncytial common ancestor, intermediate forms have persisted to the present throughout the eukaryotic tree as syncytia but have so far gone unrecognized.",
"conclusion": "Conclusion Unlike prokaryotes, eukaryotes have complex systems of intracellular membrane flux and possess organelles. They are in terms of morphology the most diverse domain of life and originated via the origin of mitochondria. Eukaryote origin is usually depicted as a narrative of two-cells-becoming-one , a one-on-one-model , where an archaeon host engulfed a proteobacterial symbiont, with the units of selection being chimeric, mononucleate, free-living cells. Our results however suggest that at eukaryote origin, nuclei, and mitochondria were the units of selection and the units of evolution within the confines of a syncytial LECA. Ancestral character state reconstruction based on taxon rich sampling spanning all supergroups suggest that LECA was 1) mitochondriate, 2) multinucleate (syncytial, coenocytic), 3) haploid, 4) with closed nuclear division, and 5) with sexual reproduction. It is often stated, also in many papers by the present authors, that the prokaryote to eukaryote transition left no intermediate forms. However, if our current thoughts are roughly on target, syncytia are in fact the intermediate state in the prokaryote to eukaryote transition, though hitherto unrecognized as such. In that light, the syncytia present throughout all eukaryote supergroups may harbor previously unrecognized forms of evidence about eukaryote origin and the prokaryote to eukaryote transition.",
"introduction": "Introduction With more than 2 million described species, eukaryotes are morphologically the most diverse domain of life ( Archibald et al. 2017 ; Adl et al. 2019 ), inhabiting a wide range of ecological habitats ( López‐García et al. 2007 ; Mora et al. 2011 ; Geisen et al. 2017 ). Eukaryotic cells are vastly more complex than prokaryotic cells as evident by their endomembrane system ( Gould et al. 2016 ). They appear about 2 billion years later in the fossil record than prokaryotes do ( Javaux et al. 2001 ; Javaux and Lepot 2018 ). There is a consensus among specialists that eukaryotes arose from prokaryotes, but the issue of how they arose from prokaryotes is intensely debated. All current theories for the origin of eukaryotes entail in some manner the concept of symbiogenesis ( Mereschkowsky 1910 ; english translation in Kowallik and Martin 2021 ) because mitochondria trace to before the last eukaryote common ancestor LECA ( Embley and Martin 2006 ; Tria et al. 2021 ) and there is no tenable way to explain the structure, DNA, and bioenergetic properties of mitochondria (and chloroplasts) without their endosymbiotic origin. The differences among current theories for eukaryote origin (reviewed in Martin et al. 2015 ; López-García and Moreira 2015 ; Dacks et al. 2016 ) mainly concern assumptions about the biological nature and cellular complexity of the host that acquired the mitochondrion. In symbiogenic theories, the host is assumed to be a typical archaeon in terms of its cellular complexity, with the origin of mitochondria precipitating genetic, cell biological and bioenergetic changes within the host-symbiont consortium that ultimately led to LECA ( Martin and Müller 1998 ; Lane and Martin 2012 ; Gould et al. 2016 ; Imachi et al. 2020 ). In gradualist theories, the host is assumed to be a descendant of the archaeal lineage, one that had however passed the threshold from prokaryotic to eukaryotic cell complexity by evolutionary mechanisms other than symbiosis, thereby bridging the gap between prokaryotic and eukaryotic complexity ( Martijn and Ettema 2013 ; Spang et al. 2015 ) before the origin of mitochondria, which therefore had little impact on eukaryote complexity. In hybrid theories, the prokaryote to eukaryote transition involved one or more additional symbioses that preceded the origin of mitochondria, such as flagella (Sagan 1967), peroxisomes ( de Duve 1969 ), the nucleus ( López-García and Moreira 2020 ), or the ER ( Gupta and Golding, 1996 ), or was precipitated by lateral gene transfer (LGT) to the host lineage, such that many hallmark traits of eukaryotes stem from genes that were invented in foreign lineages and donated to LECA via LGT ( Pittis and Gabaldón 2016 ; Vosseberg et al. 2021 ) although the methods underpinning such claims have been called into question ( Martin et al., 2017a ; Tria et al. 2021 ; Nagies et al., 2020 ). Gradualist and hybrid theories typically posit an origin of phagotrophic feeding within the archaeal host lineage before the origin of mitochondria ( Doolittle, 1998 ; Spang et al., 2015 ; Zaremba-Niedzwiedzka et al., 2017 ; Vosseberg et al. 2021 ), which is however a deeply problematic proposition from the physiological standpoint ( Martin et al. 2017b ) and at odds with evidence from the microfossil record indicating a late origin of phagocytosis ( Mills, 2020 ). Eukaryotes are unquestionably genetic chimeras, with the majority of eukaryotic genes stemming from bacteria rather than archaea ( Brueckner and Martin 2020) , wherein the bacterial genes in eukaryotes trace to LECA, not to lineage-specific acquisitions during eukaryotic evolution ( Nagies et al., 2020 ). Despite their diversity and differing underlying premises, theories for eukaryote origin uniformly entail the assumption, usually implicit, that LECA was unicellular and mononucleate ( Gould and Dring 1979 ; Cavalier-Smith 1987 ; Lake and Rivera 1994 ; Gupta and Golding 1996 ; Horiike et al., 2004 ; Imachi et al., 2020 ; Martijn and Ettema, 2013 ; Martin et al., 2015 ), an assumption that has almost never been called into question ( Garg and Martin 2016 ). The uniformity of thought on the mononucleate nature of LECA is so pervasive that it is taken as a given, that is, it is rarely, if ever, even mentioned as an assumption. More tellingly, theories for eukaryote origin, if they are illustrated with a schematic diagram at all, invariably convey an image of LECA as a mononucleate cell. Such images are often symbolic in nature, depicting traits as opposed to living cells, but at the same time, they influence the way we conceptualize the problem of eukaryote origin. Models for eukaryogenesis that involve mitochondria in a mechanistic role usually entail one-to-one relationships or many-to-one relationships ( Lane and Martin 2012 ) between mitochondria and the nucleus, whereby the nature of LECA’s nuclear dynamics, heterogeneity among nuclei in LECA, its coordination of nuclear division with cell division, its cell cycle (meiotic vs. mitotic) and the evolutionary sequence linking organelle division, nuclear division, and cell division are seldom discussed ( Cavalier-Smith 2010 ; Garg and Martin 2016 ). Why is the possibility of a multinucleated state for LECA of interest? The main evolutionary benefit that a multinucleated state would confer upon LECA is evident: Gene mutations or even severe chromosome mutations, including aneuploidies that would otherwise be lethal in a mononucleated cell could be complemented by mRNA from other nuclei in the same cytosol, permitting the survival of the (multinucleated) individual as a collection of heterogeneous nuclei, a stable starting point from which the myriad differences between prokaryotic and eukaryotic chromosome segregation and handling across cell divisions could evolve ( Garg and Martin 2016 ). In this way, the multinucleated state would buffer the transition from prokaryotic to eukaryotic chromosome division and furthermore decouple it from the evolutionary hurdle of surmounting the transition from prokaryotic to eukaryotic cell division as well as prokaryotic to eukaryotic chromatin organization during the cell cycle ( Brunk and Martin 2019 ). The occurrence of multinucleated taxa has been reported in members of all eukaryotic supergroups and in numerous higher taxa, some ancient and some derived ( Archibald et al. 2017 ; Adl et al. 2019 ; see supplementary table 3 , Supplementary Material online). Well-known examples of multinucleated forms occur within the amoebozoan supergroup: the myxomycetes (myxogastrid amoebae), protosporangiids, dictyostelids, vampyrellids, and schizoplasmodids ( fig. 1 ). Fungi are perhaps the most common coenocytes on Earth, wherein most of the classes and orders have multinucleated representatives, with unicellular forms being generally rare and often secondarily derived ( Kiss et al. 2019 ). Besides fungi, within opisthokonts, nuclearid amoebae ( Dirren and Posch 2016 ) and ichthyosporeans are also multinucleated, and syncytia are very well known among animals, for example, the body of hexactinellid sponges ( Leys 2003 ), the muscles of all the other animals, and the larvae of holometabolous insects including Drosophila . Moreover, it has long been proposed that the common ancestor of Metazoa could have been multinucleated ( Hadži 1953 ). Within Rhizaria, the deepest branch in SAR, there are numerous examples of multinucleated representatives (the most remarkable being Xenophyophorea). Furthermore, Opalinata and Apicomplexa have multinucleated forms as part of their life cycles as well ( Archibald et al. 2017 ; Adl et al. 2019 ). Not only are syncytia found among heterotrophic eukaryotes but there are also numerous examples of multinucleate algae, both red ( Florideophyceae ) and green ( Ulvophyceae ), as well as various multinucleated tissues in land plants ( Niklas et al. 2013 ). Multinucleated forms also occur among eukaryotes with secondary plastids such as in Chlorarachniophyceae , Phaeophyceae and Xanthophyceae ( Niklas et al. 2013 ). The distribution and evolution of multinucleate tissues among eukaryotes with plastids reveal a great variety of form across 60 archaeplastid families and five diverse algal lineages ( Niklas et al. 2013 ). Fig. 1. Representation of the diversity of the groups harboring multinucleated representatives. (A) Foraminifera: Filosa, a deep sea coenocytic xenophyophore; (B) Endomyxa: Lateromyxa gallica , multinucleated predatory amoeba; (C) Tubulinea: Chaos sp. multinucleated amoeba; (D) Mesomycetozoea: Sphaeroforma arctica , coenocyte with blue nuclei; (E) Protostomia: Drosophila melanogaster , multinucleated embryo; (F) Hexactinellida: Euplectella aspergillum coenocytic hexactinellid sponge; (G) Deuterostomia: multinucleated mouse muscle cells; (H) Heterolobosea: Acrasis rosea , fruiting body; (I) Chloroplastida: Ulvophyceae: Cladophora sp. syphonous thallus; (J)—Rhodophyta: Florideophyceae: Lithophyllum sp.; (K)—Myxomycetes: Multinucleated plasmodium of a Physaraceae member; (L) Ascomycota: Eremothecium gossypii , aseptate hyphae. Photo credits and Creative Commons (CC) sharing domain: A and F. NOAA, public domain; B. Norbert Hülsmann, BY-NC-SA 2.0; C. and I. Proyecto Agua, BY-NC-SA 2.0; D. Multicellgenome lab, BY 2.0; E. Billy Liar, BY-NC-SA 2.0; G. Kevin A. Murach, NIH Image Gallery, BY-CN 2.0; H. Shirley Chio, Biology of Fungi Lab, UC Berkeley, California, BY-SA 3.0; J. Christophe Quintin, BY-NC 2.0; K. André Amaral, distributed under CC BY-NC 4.0; L. Jaspersen Lab, public domain. Scale bar is approximate. Some researchers distinguish between the terms syncytium and coenocyte based on the mechanism underlying the multinucleated state, with syncytia arising from cell fusions and coenocytes arising from chromosome segregation and nuclear divisions, without cytokinesis ( Daubenmire 1936 ). Both lead to a multinucleated state and they are not mutually exclusive. We use the term multinucleated to describe the condition of having more than two (usually four or more) nuclei in the same cell without regard to the mechanism that gave rise to that state. Standard mitotic and meiotic intermediates are, obviously, not scored here as multinucleated states here, as this would trivialize the trait, making it as universal as the presence of nuclei themselves. The images in figure 1 convey an impression of a multinucleated state in the sense intended in this article. The foregoing observations lead to the question of how far back in eukaryote evolution the syncytial state can be traced. Are multinucleated forms across all eukaryotic supergroups the result of convergence or do they reflect an ancestral state? Here, we explore the presence of multinucleated forms across the breadth of eukaryotic diversity, the likelihood of a multinucleated syncytial LECA using ancestral state reconstruction and the consequences for LECA’s lifestyle.",
"discussion": "Discussion The origin of eukaryotes was a unique event from which all the complex life stems. The symbiosis that gave rise to eukaryotes occurred over 1.5 billion years ago ( Knoll et al. 2006 ). While eukaryote origin cannot be forced to occur in the laboratory, endosymbiosis can ( Mehta et al. 2018 ). The contours of eukaryogenesis, intermediate stages, and the sequence of events involved can be addressed via inference from the comparative investigation of modern lineages. The first eukaryote was the result of interactions between archaea and bacteria, two highly divergent cell lineages, that gave rise via interaction and cooperation to a new kind of organism, LECA, with new properties, novel bioenergetics, chimeric chromosomes, a cell cycle, novel genetics, reciprocal recombination, and cellular complexity. Descendants of these symbiotic partners are preserved as bacterial ribosomes in mitochondria and archaeal ribosomes in the eukaryotic cytosol. LECA had sexual reproduction that included the fusion of haploid nuclei selected for the reproduction (gametes) and the recombination of their genetic material (meiosis). Mitochondria, sex, and multiple nuclei are signatures of LECA’s state, with synergistic interactions. Unlike mitochondria, the nucleus has a large, complex genome with little size constraint. The genetic compatibility of nuclei and mitochondria inhabiting the same cytoplasm is crucial for the survival of eukaryotic cells. Internal competition or cytonuclear incompatibility can be lethal ( Blackstone and Green 1999 ; Pesole et al. 2012 ; Rand and Mossman 2020) or render the organism dysfunctional. Inheritance of mitochondria is often uniparental. The inheritance of the nuclear genome is, however, bi-, tri-, or multi-parental. Uniparental inheritance of mitochondria indicates the existence of strict control on compatibility. Meiotic recombination, ancestrally during the zygote phase, is a compatibility checkpoint. At the onset of eukaryote evolution, the compatibility of mitochondria with newly arisen nuclei was essential. In mononucleate cells, only compatible combinations survived natural selection. In syncytia, many-to-many interactions among mitochondria and nuclei buffered compatibility within the environmental confines of a single cytoplasm. Spores spawned from a syncytial LECA presented a powerful bottleneck of selection for cytonuclear compatibility ( Garg and Martin 2016 ). An intriguing aspect of the multinucleated state for LECA concerns the transition from prokaryotic to eukaryotic chromosome segregation. In prokaryotes, chromosome segregation is linked to cell division via chromosome attachment to the cell wall. In eukaryotes, microtubule-dependent segregation of condensed chromosomes and cell division (cytokinesis) are neither physically nor mechanistically linked, though often temporally apposed. That is, chromosomes can, and often do, replicate and segregate in nondividing cells without the formation of spindles for the division of the nucleus itself ( Geitler 1953 ), processes that were termed Amitose in the older literature ( Strasburger 1908 ). If the origin of nuclear division (replication followed by segregation) preceded the origin of cell division at eukaryote origin (the converse could hardly be true), the resulting syncytium need not have possessed well-regulated chromosome segregation at the outset. It could have generated nuclei with aberrant chromosome numbers or aneuploid haploids. Such defective nuclei would be lethal for a mononucleate cell, but not in a syncytium, because even highly defective nuclei could complement each other freely via mRNA in the cytosol. The multinucleate state would thus buffer virtually all deleterious effects of nuclei arising as products of incorrect chromosome partitioning during a closed protomitosis at the origin of eukaryote chromosome segregation. This would have kept the syncytium as a unit of vegetative proliferation alive, while harboring nuclei with very different chromosome sets, nuclei that kept each other viable within the syncytium through complementation via mRNA in the cytosol. This involvement of ribosomes, whose synthesis requires massive rRNA gene expression, for complementation would explain why the nucleus: cytoplasm volume ratio ( Kern-Plasmarelation ) tends to approach a roughly constant value ( Klieneberger 1917 ) of 1:10 even in syncytial cells ( Sitte et al. 1991 ). As Strasburger (1908) put it: “In the Characeae, amitotic nuclear division in internodial cells is not a degenerate process, rather it is a means to amplify certain components of nuclear substance in relationship to the increase of cytoplasmic mass” (p. 40, translation by the authors). Physical fusion of nuclei, a primitive and unregulated forerunner of karyogamy (present in LECA because LECA had sex), would generate new combinations of chromosomes at the same time as genes were being transferred from mitochondria to the nuclei ( Lane and Martin 2012 ; Garg and Martin 2016 ). That generated a heterogeneous population of nuclei interreacting with a heterogeneous population of mitochondria, within the same syncytium. A syncytium could also become physically severed, generating segments or fragments that, provided means of sealing off ends, could have generated descendant progeny (as diaspores) without the requirement for regulated cell division. Syncytial fragments provided a mechanism for propagating populations of nuclei and mitochondria. But the main evolutionary hurdle to be crossed was evolution of regulated, symmetric chromosome segregation that took into account the nutritional state of the cell ( Brunk and Martin 2019 ) en route to a cell cycle—the backbone of eukaryotic cell biology. Within a syncytium, both nuclei and mitochondria were units of selection and units of evolution. They were the intermediate state in the prokaryote to eukaryote transition. They coexisted within the same cytosol. Nuclei became heritable collections of genes able to influence their immediately surrounding cytosol, and able to interact with each other and with mitochondria via exported mRNA. Multinucleated cells are ubiquitous among the eukaryotes, both living ( figs. 1 and 3 A ) and fossil, such as a recently reported 1-billion-year-old coenocytic green alga ( Tang et al. 2020 ). Conflict and Co-operation in a Syncytial LECA Mitonuclear compatibility is important and is proportional to cell fitness ( Rand and Mossman 2020) . To compare the relative fitness of a mononucleated cell (monokaryon) and a multinucleated cell (polykaryon), one can consider the difference between the probability of survival for a population of unicellular mononucleate eukaryotes versus that for a single syncytium. For monokaryons, the probability of survival of the population is dependent on the individual survival probabilities which in turn depend on the fitness of the respective mitonuclear pair. However, in the case of a syncytium since the mitochondria and nuclei coexist in one cell the survival probability depends on the cumulative fitness of all possible combinations of mitonuclear pairs. This in turn allows the syncytium to behave similar to a population while allowing selection to resolve internal mito-nuclear conflicts independently. This is schematically shown in figure 4 and mathematically described in supplementary information 11, Supplementary Material online. A syncytium behaves as more like a population of nuclei and mitochondria than as an individual cell. Thus, the syncytium has a higher chance of survival than a population of monokaryons. Of course, there are ancient lineages of eukaryotes harboring mononucleate forms, including the excavates. However, a multinucleated LECA explains why modern eukaryote diversity is more readily derived from a syncytial ancestor than from a population of mononucleate unicellular ancestors (monokaryons). A population of monokaryons, especially that of haploid monokaryons, is not likely to accumulate genetic diversity. A syncytium on the other hand, easily accumulates genetic diversity within one cytosol, as nuclei with advantageous alleles complement deficiencies of other nuclei, and karyogamy, of which a meiotic LECA was capable, within a syncytium can generate novel chromosome combinations ( fig. 4 ). Fig. 4. Syncytia buffer chromosome defects, unlike monokaryons. Schematic representation of a population of unicellular protists and a syncytial cell. Genomes of each nucleus are schematically shown as grey lines, deficient alleles as red rectangles whereas the beneficial allele is shown in green. If the same evolutionary constraints are applied, monokaryons’ population is more likely to go extinct than a syncytium, as nuclei from different cells can neither cover each other’s defects nor buffer mitonuclear incompatibilities, while in syncytium they can. Evolutionary transitions in individuality involve cooperation and conflict ( Buss 1987 ; Maynard Smith and Szathmáry 1996; Michod 1999 ). Without mechanisms for conflict mediation, cooperation cannot survive ( Nowak 2006 ) and the higher-level unit cannot emerge ( Radzvilavicius and Blackstone 2018 ). In evolution, the population structure has always been recognized as one of the most general mechanisms favoring cooperation. Even if selection favors non-cooperating defectors, as is typically the case, cooperation might still evolve in a structured population. Consider a population made up of individuals (the lower level) divided into groups (the higher level). While defectors are favored at the lower level, cooperators are favored at the higher. If a population was one large group, the selection at the higher level is weak, and defectors prevail. In a population with many small groups, however, the selection is potentiated at the higher. Groups of cooperators can form by chance and outcompete groups of defectors ( Szathmáry and Demeter 1987 ). Thus, larger groups (e.g., a syncytial LECA) invite more conflict, while smaller groups (particularly sexually produced gametes) entail less. With larger cell sizes, stochastic processes may hence have been less important in mediating evolutionary conflict. Origins of Flagellated Eukaryotes In comparison to prokaryotes, the eukaryotic cell cycle is as unique as the processes behind mitosis and the physical separation of the newly emerging cell (cytokinesis). While a few homologous proteins are shared between archaeal binary fission and eukaryotic cytokinesis ( Lindås et al. 2008 ), the mechanism of chromosome segregation through a centrosome-organized microtubular system and the subsequent actin-based cell constriction is not conserved across the prokaryote–eukaryote divide. The mechanism of eukaryotic chromosome segregation, like other eukaryote-specific traits, evolved de novo during the endosymbiotic integration of a bacterial partner within an archaeal cytosol en route to LECA. In a syncytium, chromosome segregation likely involves molecular selforganization of a chromosome separating machinery that requires no anchoring points at the plasma membrane. Closed mitosis, in which the nuclear envelope remains largely intact, is considered ancestral to open mitosis ( Cavalier-Smith 2010 ), consistent with our own results ( fig. 3 ). All variants of mitosis share a microtubule-based network, which can be bundled or loose in a star-like manner, that reach out for the chromosomes and attach at the kinetochore which was present in LECA ( Tromer et al. 2019 ). Centrosomes are however, not essential for chromosome separation ( Heald et al. 1996 ). Crucially, eukaryotic chromosomes are separated largely by pushing forces along microtubules, in which the eukaryote-specific kinesin family of proteins play an essential role ( Shimamoto et al. 2015 ). These mechanisms fit seamlessly with the biology of a syncytial cell, as mitosis of individual nuclei can occur independently of localized plasma membrane fixation points. Consequently, the origin of mononucleated, flagellated protists can be viewed from a novel perspective. Images of the closest living relative of the archaeal host cell and a bacterial partner depict two sessile, nonmotile partners ( Imachi et al. 2020 ), the syncytial LECA we propose was sessile, too. The microtubule organizing center (MTOC), or basal body, and the ability to form flagella was present in LECA. This trait diversified among eukaryotic supergroups and underwent recurrent loss ( Yubuki and Leander 2013 ). Eukaryotic flagella are directly connected to basal bodies, or they form in a centriole-dependent manner de novo ( Schrøder et al. 2011 ). The flagella pore complex shares a number of proteins with the nuclear pore complex ( Dishinger et al. 2010 ; Kee et al. 2012 ; Gould et al. 2016 ). We suggest that the flagellum evolved on the basis of a (duplicated) centrosome-derived structure that subtended a region of the plasma membrane. Mononucleate, flagellated spores could have thus emerged from the syncytium with the actin cytoskeleton supporting final scission ( Heidstra 2007 ). Only spores containing viable mitonuclear interactions and capable of flagellar motion would have had the properties of motile gametes, provided that they were able to fuse with others of their kind, which is possible given the tendency of archaea themselves to fuse ( Lange et al. 2011 ; Garg and Martin 2016 ; Shalev et al. 2017 ). Such spores would present motile units of selection. The nucleus of many flagellated protists is located in close proximity to the basal body, if not connected to it, as in numerous Archamoebea, Chytridiomycota, Olpidium , Pelagophyceae, Bacillariophyceae, Rhizaria and others (reviewed in ref. 2). It is possible that such gamete like cells became the founders of eukaryotic supergroups, all of which contain flagellated representatives that can generate syncytia ( fig. 3 A ). We have no suggestion for the physical size of LECA as a syncytium, although we do suggest that it was a marine sediment dweller ( Martin and Müller 1998 ), where anaerobic syntrophy is essential to symbiotic interactions ( Imachi et al. 2020 ). The hyphae of modern fungal individuals can cover areas of square miles ( Anderson et al. 2018 ). LECA could have been a large non-dividing multinucleate unicell that spawned supergroups through the extrusion of mitochondriate flagellated spores. A 6-min animated video illustrating the origin of eukaryotes from symbiosis and the role of a syncytial state in the life cycle of LECA can be viewed at ( https://www.youtube.com/watch?v=mmh_IpdgWvw&t=2s )"
} | 7,233 |
39081113 | PMC11289642 | pmc | 4,133 | {
"abstract": "Central place foragers, such as many ants, exploit the environment around their nest. The extent of their foraging range is a function of individual movement, but how the movement patterns of large numbers of foragers result in an emergent colony foraging range remains unclear. Here, we introduce a random walk model with stochastic resetting to depict the movements of searching ants. Stochastic resetting refers to spatially resetting at random times the position of agents to a given location, here the nest of searching ants. We investigate the effect of a range of resetting mechanisms and compare the macroscopic predictions of our model to laboratory and field data. We find that all returning mechanisms very robustly ensure that scouts exploring the surroundings of a nest will be exponentially distributed with distance from the nest. We also find that a decreasing probability for searching ants to return to their nest is compatible with empirical data, resulting in scouts going further away from the nest as the number of foraging trips increases. Our findings highlight the importance of resetting random walk models for depicting the movements of central place foragers and nurture novel questions regarding the searching behaviour of ants.",
"introduction": "1 . \n Introduction Animals that carry resources back to a particular site, such as their nest, are called central place foragers. The decisions of central place foragers are affected by external factors such as food quality or distance to food patches [ 1 – 4 ]. The mechanistic understanding of the movements of animals in general and of central place foragers in particular concerns the relationship between the internal state of the animal, its locomotion and navigation abilities and the environmental context (e.g. distribution of resources) [ 5 , 6 ]. The feedback loop between food patch characteristics and foraging behaviour, driven by the ability to locate food, results in animals exhibiting various movement patterns, including random walks [ 7 , 8 ] and Lévy walks [ 9 – 11 ]. From the movement, behaviour of animals (microscopic scale) emerges a foraging area or a territory (macroscopic scale), which can be mechanistically inferred from the movements and behaviours of individuals [ 12 – 14 ]. Such a mechanistic understanding of territory or home range formation in animals is instrumental to predict the impact of environmental perturbations on the survival and reproduction abilities of a species [ 14 – 16 ]. Social insects, including ants, provide a great opportunity to study phenomena that emerge from self-organization [ 17 ]. For instance, the potentially large number of workers in a social insect colony ensures that the macroscopic scale emerging from the multiple interactions at the microscopic scale has a spatial and temporal relevance for explaining colony-level decisions and behaviours. This is particularly true for the emergence of a foraging area or a territory from individual movements [ 18 , 19 ], such as in colonies of social bees, where the scouting movements of workers can be used to understand the complex dynamics between pollinators and plant reproduction [ 20 ]. Many ant species are central place foragers, living in a nest and exploiting the surrounding environment. It is still unclear, however, how the behavioural mechanisms of searching ants, including their movements and the timing of their decisions, result in the exploited foraging area of a colony. More specifically, a modelling framework unifying known behavioural mechanisms involved in searching for ants with a macroscopic description of the foraging area at the colony level is lacking. The movements of single ants navigating a simple laboratory environment can be described in fine detail by a correlated random walk [ 21 ], although other movement types such as meandering may also be present [ 22 ]. At the individual scale, this behaviour results in a series of straight moves of varying lengths, separated by turning angles favouring new directions close to the previous ones, meaning that the ants have a tendency to make small deviations at each reorientation event. At the collective scale, the description of a population of ants moving with a correlated random walk corresponds to a diffusive pattern. For a given ant nest, if ants whose task is to search the surroundings to find resources—so-called scouts—move with a correlated random walk, this would result in all scouts, on average, progressively going away from their nest and their density decreasing over time in areas close to the nest. In other words, this would result in an unrealistic outcome of scouts moving away from the nest, presumably until they find resources, and the density of scouts being homogeneously very low around the nest. This is unrealistic because the time to find resources may exceed the time a scout can survive outside its nest, and the energy costs associated with such an exploration pattern would be very high at the colony level. By investigating the behaviour of ants on their inbound journey, returning to their nest and searching for a nest entrance, studies have revealed a looping behaviour, where individuals go back and forth from their initial location, gradually increasing the sizes of the loops [ 23 – 26 ], in agreement with a looping behaviour reported in fire ants searching for food [ 27 ]. This searching behaviour of foraging ants may therefore well be involved in outbound journeys, when foragers are exploring the environment without individual spatial preferences or when they are not responding to recruitment [ 28 ]. This searching strategy involves sinuous random walk outbound movements and straighter trajectories back to the nest [ 28 ]. The searching behaviour of ants has been investigated theoretically with models developed at the individual scale, but with little consideration for the associated emerging collective pattern, namely, the foraging area [ 22 , 23 , 29 – 31 ]. There are methods to estimate, in the field, the foraging area and territory of an ant colony, based on foraging or aggressiveness responses and nest dispersion [ 32 – 34 ]. Theoretical models, based on the same principles, have also been proposed to estimate the size and arrangements of the territories of ant populations [ 35 , 36 ]. These methods, theoretical and empirical alike, often reveal, infer or predict the general shape of the borders of a territory (e.g. circular [ 32 ]) at a given time, but do not provide a mechanistic and dynamic description of its emergence [ 14 ]. It is, however, worth noting a few quantitative properties of searching and foraging ants at the macroscopic scale that help to characterize the foraging area. In ants searching for their nest entrance in an unknown environment, several studies on various species (the Australian desert ant Melophorus bagoti and a western Mediterranean ant Aphaenogaster senilis ) have reported that the distribution of the distance between individuals and their search origin can be depicted by two exponential distributions [ 25 , 28 ]. This suggests that there is a general mechanism in searching ants moving around a central place that gives rise to ant displacements being exponentially distributed—therefore indicating that the probability of finding resources decreases exponentially with the distance from the nest. In fire ants, it was found that the time spent by scouts outside their nest before returning was distributed with a power law [ 27 ]. In other words, the probability for a single scout to return to the nest was not found to be constant in time: the longer the individual spent outside the nest, the lower the probability of returning (compatible with the qualitative reports of increasing loops exhibited by searching desert ants). In a recent study focusing on the emergence of polydomous networks in red wood ants Formica lugubris , we made the assumption that scouts have a tendency to return to their nest even if they are unsuccessful in finding resources and predicted that the resulting distribution of scouts around their nest would be exponential [ 4 ]. It has not, however, been thoroughly shown how the individual mechanisms involved in searching behaviours previously reported in the literature are connected with the macroscopic description and the range of the foraging area [ 37 ]. Here, we aim to develop a modelling framework that will unify both the individual and collective descriptions of scouting ants. We formulate a spatially explicit model, extending a correlated random walk model with a tendency for ants to stochastically return to their nest. Such models, depicting random walk movements with stochastic returns to a central place have recently received attention in statistical physics, leading to an emerging literature on random walks with stochastic resetting [ 38 ]. This framework is well suited to describe central place foraging [ 20 , 39 , 40 ]: it allows description of the system at both microscopic and macroscopic scales and the analysis of the effects of varying both movement patterns and the dynamics of the returning (resetting) episodes [ 41 , 42 ]. This class of models has already been used to investigate the foraging movements of honey bees [ 20 , 39 ], where it was suggested that the rate of returning to the nest was constant in time [ 20 ], similar to what has been proposed in ants [ 4 ]. Here, we extend a random walk model for ant movements [ 21 ] with a stochastic resetting process to evaluate whether or not the hypothesis of a nest-returning rate that is constant in time for unsuccessful foragers is compatible with empirical data at both the individual and collective levels. We specifically explore how constant, linearly decreasing or exponentially decreasing rates to return to the nest with respect to time determine the duration of journeys outside the nest and the distribution of scout distances from their nest. We evaluate these alternatives by reanalysing an existing dataset of searching ants and using published results reported in various ant species, in both laboratory and field settings. We suggest important metrics for discriminating different models of random walks with resetting mechanisms and discuss the implications of our findings for understanding how the exploration range of an ant colony is derived from simple movement rules, and what may affect resets in scouting ants.",
"discussion": "4 . \n Discussion In this article, we propose a new model depicting the movements of searching and scouting ants, made of a discrete correlated random walk model and of a stochastic returning mechanism to the nest in the event of unsuccessful search. The main result of this study is that such a returning mechanism very robustly ensures that scouts exploring the surroundings of a nest will be exponentially distributed with distance from the nest. In other words, scouts will explore more efficiently the immediate surroundings of the nest than regions further away, no matter the actual rules governing their movements or their tendency to return to their nest. In bees, it was recently proposed that individuals would have such a returning mechanism, but with a probability of returning to the nest constant in time [ 20 ]. We show here, for a different movement pattern made of series of straight lines, that whether this probability is constant with the searching time or not actually does not change the shape of the emerging distribution of distance of scouts from their nest of origin. However, we stress here a major difference: when the probability of returning to the nest is not constant in time, the emerging distribution is no longer stationary, up to a point where the probability reaches its minimum value. Our study further shows that, in ants, this probability to return to the nest is not constant with the number of reorientation events. We first show, analysing empirical data from a laboratory experiment of single T. albipennis ants searching a foraging arena, that the distance at which individuals initiate inbound journeys (homing) increases with time, leading to loops of increasing radius away from the nest, which is not compatible with a model using a probability of returning that is constant over time. An additional evidence of these increasing loops is shown by the survival curve of searching bouts, which is not exponentially distributed but instead decreases as a power law in the empirical data with T. albipennis ants that we analysed, in contrast to the model in which the probability of returning is constant. The fact that the survival curve of searching bouts was decreasing as a power law had already been reported in data collected in the field on fire ants [ 27 ]. The finding that searching ants make increasing loops around their initial location resonates with a behaviour reported in the field for desert ants of the genus Cataglyphis and M. bagoti when searching for their nest entrance [ 23 , 25 ]. In the Australian desert ant, it was also reported that the average distribution of the distance of searching ants from their initial location was made of two distinct exponential distributions in unfamiliar environments [ 25 , 26 ], very similar to what we show in T. albipennis . The presence of two distinct exponential distributions was also reported in laboratory conditions in the ant A. senilis [ 28 ]. To explain these two exponential distributions, authors suggested the presence of two searching modes, either by the means of two distinct random walks [ 25 ] or with risk-prone and risk-averse strategies [ 28 ]. Here, our model and empirical data analysis suggest a new explanation: there is only one searching mode but the probability of returning to the nest not being constant in time leads the distribution of distance to be made of two distinct exponential distributions once averaged across all ants. It is worth noting here that in our model the emergence of two exponential distributions appears only when averaging across all number of reorientation events. In other words, the two distributions are visible only when agents are not synchronized in their probability of returning to the nest—i.e. when the population under consideration is made of agents whose probabilities of returning to the nest are not the same. To conclude, we have multiple sources of evidence in searching and scouting ants of various species with different resource exploitation strategies (fire ants, ants from the genus Cataglyphis , Australian desert ants M. bagoti , T. albipennis and Ap. senilis ), in both field and laboratory conditions, of a returning mechanism made of increasing loops. Our simple model, made of a correlated random walk and stochastic returns to the nest (spatial resets) whose frequency decreases with time, predicts all the features reported in the literature previously and in the empirical data analysed for the purpose of our study—namely, that searching ants are distributed around their nest following a distribution made of two exponential distributions and a survival curve of searching bouts distributed as a power law. This highlights the relevance of random walk with stochastic resetting models to describe ant-searching movements at both the individual and collective or colony levels. Regarding the functions of the mechanisms that we found, this exponential distribution of distances may be a key mechanism at the colony level to ensure that the close vicinity of the nests is more crowded than areas further away, improving defence and the ability of individuals to quickly alert the other nest members of any danger located at short distances from the nest [ 45 ]. At the collective level, the emerging spatial pattern of scouts around their nest, exponentially decreasing with distance to the nest, may therefore be the result of a trade-off between exploration of the environment and safety of the nest—and of the individuals. It may be that the simple mechanism of having a probability to return to the nest that decreases with time allows for the emergence of a composite search model, in line with theoretical predictions that showed that foraging bouts made of an intensive search phase, followed by an extensive phase if no food is found in the intensive phase, are efficient [ 46 , 47 ]. However, the fact that we found that any movement mechanism combined with a returning mechanism will lead to an exponential distribution of distances questions whether this is an adaptive emerging pattern or rather a mandatory byproduct of central place foraging in highly social species. Even if the general shape of the exponential distribution might not be adaptive, its actual parametrization in different species might be. Different movement behaviours or returning rules may well indeed be finely tuned so that the stationary distribution of scouts around the nest is well suited to the needs and objectives of the colony. Our model provides predictions at the nest level while mainly requiring calibration from individual trajectories in simple and non-expensive laboratory experimental apparatuses. Our study suggests metrics at both collective and individual levels that may be suitable to investigate further the actual behavioural rules of movements and of returning used by different ant species, in relation to the pattern of scout distribution and exploration at the colony level. This avenue of research, in addition to shedding light on scouting behaviours and spatial patterns in social insects, may be of interest to improve estimates of the foraging area of a colony in the field, from simple laboratory experiments. Such estimates are indeed very costly and difficult to get in the field, requiring practitioners to track individuals and identify their colony of origin in complex environments. Our results regarding the density of scouts and defining a stationary foraging area are important for instance to understand better how colonies exploit resources in their environment—including the specific case of polydomous ants. Polydomous ants are ant colonies distributed across multiple spatially separated nests connected to each other and to foraging patches by a network of trails [ 48 ]. Predicting how scouts are distributed around their nest and, therefore, inferring the probability of finding resources whether they lie within or outside the foraging area established by scouts with a returning mechanism is instrumental to understand the costs and benefits of polydomy [ 49 , 50 ] and the emergence of trail networks and polydomous networks, which are the results of colonies solving a quality–distance trade-off [ 4 ]. Our simple model abstracted several details of ant locomotion, for instance as found in the species T. albipennis that was used in the empirical analysis we reported. We found a complex interaction between speed and sinuosity that may depend on the journey duration or distance from the nest. In our model, agents walk at a constant speed and return to the nest ballistically. While we showed that our simple model could still account for all emerging patterns and results, both previously reported or newly analysed in this study, such movement details will be important to integrate in future research, to closely investigate the link between individual movement and the resulting colony-level distribution of workers. For instance, more complex returning movement patterns were recently incorporated in a random walk model with stochastic resetting depicting bee movements [ 20 ]. Our study, proposing a class of models with both spatial and temporal resets paves the way to investigate novel questions in the future. First, regarding the translation of the movement behaviours measured in the laboratory to estimates at the colony level in the field, it is unclear how accurately parameters estimated in laboratory experiments would describe behaviours occurring in nature. In other words, do ants adapt their returning behaviour when evolving under laboratory conditions in which their nest size and foraging area are heavily constrained or would this behaviour be finely tuned to natural conditions? Investigating this would be an important step in inferring foraging area from laboratory experiments. A second open question concerns the time scale at which the returning probability operates. Since we showed, based on empirical observations, that this probability is likely not constant with time, and decreases with the duration of the searching journeys of ants, the question of the actual clock underlying this effect is still open. Moreover, it is unknown how asynchronous is this temporal reset in the scout population. Is this clock reset at the scale of the hour, when scouting workers start a new searching session? Or at the scale of the day, between resting periods? And how does the age of the worker interfere with this probability, with older scouts maybe having a lower probability of returning on average, that would allow them to explore further away than younger ones—the colony age structure therefore affecting the foraging area? Different movements [ 22 , 31 , 51 ] or returning rules coexisting may also underlie a complex stationary distribution made of several distinct exponential distributions. These questions are important to understand better the emergence of foraging or searching areas in an unknown environment in ants and other social central place foragers."
} | 5,374 |
39984829 | PMC11846334 | pmc | 4,135 | {
"abstract": "Background Microhabitat environmental factors (e.g., temperature, oxygen concentration, nutrients, osmotic stress, and topography) are critical to the survival of intertidal organisms. Understanding how transcription responses are regulated in relation to intertidal microhabitat variation has important implications for studying adaptive evolution in these organisms. The barnacle Chthamalus challengeri , which survives in the intertidal zone and is subjected to periodic tidal changes, serves as an ideal species for detecting adaptive evolution in intertidal organisms. Results In this study, we designed a series of in situ tidal conditions for C. challengeri and sequenced their transcriptome collected from various microhabitats. We aimed to detect the genetic adaptation mechanisms of barnacles responding to the microhabitat changes in the intertidal zone based on comparative transcriptomics. Our results indicated that different intertidal microhabitats significantly affected the gene expression models of C. challengeri , particularly for genes related to physiological and biochemical functions. Specifically, the expression of genes such as CYP450, HSP70, CYTB, and COX1 was significantly increased under low tide (air-exposed conditions), while genes like CNGA3, AK, and CP52 showed significantly increased expression under high tide (seawater-immersed conditions). Conclusion The results suggest that C. challengeri relies on cytochrome p450 enzymes to enhance oxidative capacity, counts on heat shock proteins and cell phagocytosis to resist microhabitat changes in response to different tidal conditions, and produces a hypoxic stress response to regulate energy metabolism and body temperature changes upon entering into seawater. This study provides genetic resources and clues for investigating the adaptation mechanisms of intertidal barnacles and identifies different gene expression models for C. challengeri responding to various microhabitats. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11357-8.",
"conclusion": "Conclusion We report the transcriptomes of C. challengeri from various intertidal microhabitats. Through comparative analyses of atmosphere and immersed groups, we identified key genes involved in the intertidal adaptation of C. challengeri when exiting and entering water. A strong immune response and heat shock response were observed in the atmosphere group, particularly under conditions of increased temperature and water deprivation. In the immersed group, energy metabolism and thermoregulatory mechanisms exhibited rapid activation in response to hypoxic stress upon water-entry. This indicates that C. challengeri likely employs its innate immune system to continuously counteract stress responses along with its microhabitat periodical changes. These coping strategies not only highlight the resilience of C. challengeri but also provide essential insights for future studies on the adaptation mechanisms of crustaceans to intertidal environments.",
"introduction": "Introduction Surviving in the intertidal zone is full of challenges for organisms because they must withstand fluctuations in various environmental stressors, such as temperature, salinity, hypoxia, air exposure, pathogens, and variations in tidal cycles [ 1 – 3 ]. Intertidal species are thought to live close to their survival limits and are vulnerable to environmental condition change [ 4 ]. Researches on abiotic stress has demonstrated that species surviving in the intertidal zone sufficiently exhibit greater resistance to these factors compared to species living under the water [ 5 ]. Many behavioral, physiological, and genetical amendments of intertidal organisms could be observed, which are supposed to be intertidal environmental adaptation [ 6 , 7 ]. For instance, porcelain crab Petrolisthes cinctipes can tolerate air exposure for longer durations, utilizing an increased heart rate to withstand greater thermal stress [ 8 ]. Under high temperature conditions exposed to air, acorn barnacle Semibalanus balanoides may enter a state of coma, losing the ability to close their valves, which consequently decreases their survival rate, in contrast, when submerged in water, the expansion of the adductor muscles in barnacles typically accelerates, enhancing the respiratory rate in the water and speeding up their metabolic reactions [ 9 , 10 ]. In addition, the intertidal snail Echinolittorina peruviana tends to orient itself laterally towards the sun when in aggregated groups, a behavior that allows them to maintain a lower temperature during hot summer days [ 11 ]. Intertidal organisms can facultatively employ certain microhabitat selections, such as aggregation, opercular closure, and sun-oriented responses etc., to confront the harsh conditions of temperature stress, respiration, and desiccation of intertidal habitat [ 12 ]. All these indicate that the microhabitat of intertidal zone plays significant roles in shaping the physiological responses of intertidal organisms. Although previous studies have indicated that some intertidal species could regulate their behavior or physiology to resist the severe environmental conditions of tidal zone, but the researches referring to genetic regulation, which primarily focuses on the regulation of defensive genes and environmental adaptation genes, is limited [ 13 ], particularly for the intertidal sessile barnacles. Transcriptome has been used to explore the microhabitat adaption for intertidal animals, such as the upregulation of genes encoding detoxifying enzymes being widely observed under thermal stress due to air exposure in Chlorostoma funebralis , Patella vulgata , and Crassostrea virginica [ 14 – 16 ]. While for the barnacles, transcriptome was used to investigate the individual development and larval settlement in laboratorial condition for Amphibalanus Amphitrite [ 17 ], Megabalanus volcano [ 18 ], and to detect the deep-sea environmental adoption of Glyptelasma gigas through comparative transcriptome with a shallow water species Octolasmis warwicki [ 19 ]. Likewise, Yuan et al. demonstrated that the upregulation of some anti-stress factors and accumulation of acyl-carnitines are important processes for the intertidal adaptation of Capitulum mitella using transcriptome analysis [ 20 ]. Despite comparative transcriptomics has greatly beneficial in the adaption research of intertidal animals, the regulatory mechanisms of transcriptomes following the tidal variations is nearly unreported for intertidal barnacles. In this study, we sequenced the transcriptome of Chthamalus challengeri , a barnacle abundant in the internal zones of the northwest Pacific Ocean, under two different tidal conditions, the first group (atmosphere group) includes the individuals under three different exposure duration in the air, and the second group (immersed group) include the individuals under three different immersed duration in water. We used comparative transcriptomics to analyze the gene expression status of C. challengeri in different intertidal microhabitats, aiming to identify gene expression differences between the high and low tides, and further to understand its genetical responses to the microhabitat variations and adaptive mechanisms.",
"discussion": "Discussion Intertidal ecosystems, situated between the lowest and highest tide lines on shores, encompass mangroves, seagrass meadows, muddy flats, rocky shores, and sandy beaches. These areas are renowned for their high biodiversity and environmental heterogeneity [ 34 , 35 ]. Moreover, these ecosystems serve as natural laboratories for studying complex environmental stress adaptations [ 36 ]. Multiple ecological stressors, such as submersion, aerial exposure, desiccation, temperature and salinity variations, and ultraviolet radiation, impact intertidal ecosystems. Consequently, organisms inhabiting these areas must adapt to these abiotic stresses [ 37 ]. Key KEGG pathways associated with intertidal adaptation Our study found that in the air group, differentially expressed genes (DEGs) predominantly involve neuroactive ligand-receptor interactions, ECM-receptor interactions, MAPK signaling pathway, Notch signaling pathway, and Wnt signaling pathway. The entry of exogenous substances into the body initially activates ECM receptor interactions. These interactions are a core component of wound healing across all taxa [ 38 ]. Prolonged exposure to air and other stressors significantly inhibits apoptosis, which may weaken the formation of the tissue and shells of C. challengeri . Similar to other crustaceans, exogenous substances can stimulate ECM of barnacles to release stratum corneum fluid and activate innate immune responses, including the prophenoloxidase system and cell adhesion molecules [ 39 ]. This reflects hypoxic stress and changes in the immune microenvironment. Additionally, under stress from air exposure, temperature, and osmotic pressure, the Wnt signaling pathway and MAPK signaling pathway in Amphibalanus amphitrite are significantly enriched. These pathways rely on Wnt signaling to regulate cell polarity, preventing cell rupture due to excessive salinity. The MAPK signaling pathway mediates extracellular signal transduction, aiding barnacles in maintaining normal physiological functions. When exposed to air, the C. challengeri loses water, increasing internal salinity. Excessive environmental stress can lead to the accumulation of reactive oxygen species (ROS), resulting in oxidative stress and activating the innate immune system for antioxidant defense [ 40 ]. This also explains the increase in oxidative stress and cellular activities (such as adhesion and migration) in C. challengeri when exposed to air. In response to air exposure and the subsequent activation of the immune system by exogenous substances, the cellular processes of C. challengeri were significantly enriched in phagocytes and lysosomes. Cells of the mononuclear phagocyte system play a crucial role in mediating cytokinesis, wound debridement, and bridging the gap between innate and adaptive immunity [ 41 ]. Furthermore, the regulatory pathways of energy metabolism show increased metabolic capacity through amino acid metabolism, ribose metabolism, and oxidative phosphorylation, which provide energy for the organism. Gene Set Enrichment Analysis (GSEA) revealed that two P450 gene sets were significantly enriched, with P450-related genes up-regulated in the air exposure group. Additionally, glutathione transferase, involved in the ‘metabolism of xenobiotics by cytochrome P450,’ was also up-regulated in the differential expression analysis. Under air exposure conditions, barnacles accumulate substantial amounts of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals [ 42 ]. This accumulation occurs as metabolic rates increase, thereby activating the cellular stress response. CYP450 is a crucial component of the detoxification enzyme system, facilitating the excretion of metabolic waste from C. challengeri through ECM receptor interactions and ABC transporter proteins. Genes associated with intertidal adaptation Our results indicated that as the exposure temperature increased, the expression of the HSP70 in C. challengeri gradually rose. Heat shock proteins (HSPs) help protect organisms from high-temperature exposure by facilitating the folding and repair of denatured proteins. Additionally, they promote gluconeogenesis and glycogen synthesis, increasing intracellular glycogen stores to counteract stress responses [ 43 ]. As molecular chaperones, heat shock proteins are involved in coagulation reactions and the activation of defense systems in invertebrates, and they have also been shown to act as effective signals that trigger innate immune responses [ 44 ]. As intertidal rocky areas are heated due to air exposure, C. challengeri may have evolved adaptive mechanisms to thrive in these periodically elevated temperature environments. CYP450 has emerged as a significant area of research in crustaceans, with numerous studies demonstrating its critical role in resisting high temperatures in aquatic environments. CYP450 is a superfamily found in a wide variety of living organisms, including animals, plants, and microorganisms. In response to prolonged air exposure stress, the expression of CYP450 in C. challengeri is significantly upregulated. To limit heat production, CYP450 in the body may generate pyrolytic substances through monooxygenase pathways or consume necessary substrates for thermogenic reactions, such as glucose and active fatty acids [ 45 ]. Atmospheric exposure enhanced the immune activity of C. challengeri , prompting DNA methylation to regulate CYP450 and facilitate the metabolism of exogenous compounds. This adaptation may help the species cope with immune stress caused by extreme environments by activating protein hormones and defense compounds. In addition, compared to being submerged in seawater, barnacles exhibit a rapid increase in respiratory rates when emerging from the water [ 46 ]. We suppose this response is related to temperature fluctuations, as terrestrial temperatures are 4–5 °C higher than seawater temperatures. The initial oxygen consumption of Balanus nubilus in the second and third hours after water discharge may exceed 20% [ 47 ]. This also explains the upregulation of COX1 and CYTB, which are critical for electron transfer and oxidative phosphorylation in cellular respiration. Thus, we speculate that C. challengeri experiences thermal stress after emerging from the water, demonstrating an increase in energy metabolism that synergistically interacts with the previously observed activation of carbohydrate metabolism by CYP450 enzymes. When the tide rising, C. challengeri is submerged in seawater, resulting in a temperature shift from the hot rock environment to the cooler seawater. The results of our Mfuzz analysis indicate that C. challengeri showed a relatively high expression of the cyclic nucleotide-gated channel 3(CNGA3) gene in this stage. The gene begins transcription 0 h after entering the water, reaches a relatively high level at 2.5 h underwater, and then returns to normal expression levels by 5 h (Fig. 8 ). This gene integrates signals from peripheral thermoreceptors and detects chemical cues from infectious agents, coordinating physiological and behavioral thermoregulatory responses [ 48 ]. The CNGA3 promotes feedback thermoregulatory pathways through cold-sensitive activation of preoptic area (POA) neurons in the hypothalamus, regulating body temperature [ 49 ]. We predict that the high expression of the CNGA3 gene results from the sudden drop in body temperature as C. challengeri transitions from prolonged light exposure to immersion in seawater, enabling it to respond to temperature changes. On the other hand, activation of ion channel transporter proteins by CNGA3 is essential for C. challengeri to maintain osmotic pressure homeostasis in vivo. In some barnacle populations, gas bubbles fill the sleeve cavity upon water entry, leading to impaired gas exchange with flowing seawater and triggering a hypoxic stress response. Normal underwater physiology is typically restored after 1–2 h [ 50 ]. After experiencing an acute hypoxic reaction, the internal oxygen level (oxygen content in the hemolymph or mantle luminal fluid) of C. challengeri may sharply declines. AK regulates energy metabolism by catalyzing the reversible phosphorylation of arginine to produce ATP, helping to cope with the hypoxic conditions underwater. As a key enzyme in energy metabolism, it can adapt to low-temperature environments with cold permeation [ 51 ]. When the body temperature reaches the same level as the ambient temperature, its regulation returns to normal. Interestingly, within 6 h of exposure to air, the expression level of Cement protein 52 (CP52) tends to stabilize. However, after 2.5 h of immersion in seawater, we observed a significant increase in the expression level of CP52. The CP52 protein is a barnacle cement protein that typically participates in the growth of barnacles, forming a biofilm in conjunction with the secretions from the barnacle’s base [ 52 ]. Additionally, it can resist pathogen invasion in seawater through encapsulation and endocytosis [ 53 ]. This underscores the crucial role of the continuous secretion of cement proteins (CPs) by intertidal barnacles in maintaining their adhesion to substrates and defending against pathogens."
} | 4,157 |
31217664 | PMC6558992 | pmc | 4,136 | {
"abstract": "The presence of air in the anode chamber of microbial fuel cells (MFCs) might be unavoidable in some applications. This study purposely exposed the anodic biofilm to air for sustained cycles using ceramic cylindrical MFCs. A method for improving oxygen uptake at the cathode by utilising hydrogel was also trialled. MFCs only dropped by 2 mV in response to the influx of air. At higher air-flow rates (up to 1.1 L/h) after 43–45 h, power did eventually decrease because chemical oxygen demand (COD) was being consumed (up to 96% reduction), but recovered immediately with fresh feedstock, highlighting no permanent damage to the biofilm. Two months after the application of hydrogel to the cathode chamber, MFC power increased 182%, due to better contact between cathode and ceramic surface. The results suggest a novel way of improving MFC performance using hydrogels, and demonstrates the robustness of the electro-active biofilm both during and following exposure to air.",
"conclusion": "Conclusions In this study, ceramic cylindrical MFCs were used to investigate three operational challenges (i) the effect of airflow through the anode, (iii) hydraulic retention time and its effect on biofilm stability and (iii) improvement of the cathode (using hydrogel powder) i. Generally, when considering MFC operation, the user will go to lengths to limit the penetration of air into the anode chamber. This is because oxygen consumes electrons causing a decline in electrical output and it can harm strict anaerobes. We have demonstrated that the influx of air had a negligible effect on biofilm health and power output. When air was bubbling through the anode chamber the power dropped by just 1% and recovered quickly when the airflow stopped. However, the presence of air did accelerate the breakdown of organic matter and eventually at around 44 h the MFCs declined through nutrient depletion. When replenished the MFCs responded instantly demonstrating that there was no permanent damage to the biofilm. ii. Bi-directional polarisation curves highlighted the importance of hydraulic retention time on biofilm stability. The HRT of 23 h proved too long as demonstrated by a 58% drop in maximum power between first and the second polarisation sweeps. When the HRT was 4 h the MFCs performed much better on the return sweep with just a 31% drop in maximum power. We propose that the bi-directional polarisation method can be used to determine optimal operational conditions such as flow-rate. iii. When hydrogel powder was incorporated into the cathode chamber there was a 182% increase in power output after 2 months compared to zero improvement for MFCs without hydrogel. As the material swelled with the electrochemically produced catholyte, a better contact between cathode and ceramic surface was maintained alongside the production of a potentially beneficial alkaline gelatinous material.",
"introduction": "Introduction The generation and subsequent treatment of wastewater will always be a challenge to humanity. Human health and the state of the environment are under threat from pollution of the waterways and for this reason there have been significant efforts to improve treatment technologies in terms of both efficiency and cost reduction. Microbes are utilised in a number of technologies including aerobic treatment e.g. biological filters and anaerobic such as anaerobic digestion. Another technology that can complement these is the microbial fuel cell (MFC) and research efforts have grown over the past couple of decades such that there are now a number of realistic target applications. The technology can offer electricity and sanitation in remote locations as demonstrated through successful field trials using urine as fuel [30] . MFCs can be configured into systems that provide a sufficient and constant supply of energy to power useful devices such as robots [23] , meteorological buoys [42] , pumps [20] , mobile phones [13] and transceivers [32] . While these are exciting advances, perhaps the area that is most suited to the scale-up of MFCs is wastewater treatment. MFCs have demonstrated in numerous studies over the last decade that wastewater with diverse and complex compositions can be utilised as fuel [12] . The MFCs adapt and can deal with different types of waste that vary considerably in parameters such as organic loading [18] , pH [35] , conductivity [43] , sulphide content [27] , toxicity [26] and other factors. Given that the fuel can be any organic waste liquid, which is treated whilst electricity is generated, there is a real focus on scaling up the technology for wastewater treatment. Where MFCs might fit into the treatment process is still cause for debate. To date they have functioned efficiently when fed industrial strength wastewater with high organic loading i.e. of the type that might be found at the beginning of the treatment process [40] . MFCs have also shown to be suitable for operating at the end of a multi-stage process where the effluent is weaker and with low chemical oxygen demand (COD), demonstrating that they could perhaps function as a polishing stage [44] . In addition to the composition of the wastewater itself, other factors can affect MFC behaviour, potentially to the detriment of performance. For example, the flow of water will introduce oxygen and in addition, processes such as the sequential batch reactor (SBR) actively introduce air. Therefore if a MFC was to function in such an environment, it needs to demonstrate resilience to the influx of air. The SBR process is multi-stage and involves anoxic and aerobic stages and MFCs can complement the process by improving treatment efficiency when placed downstream [38] , demonstrating in this way the treatment of pharmaceutical wastewater [39] . However, while it is accepted that MFCs can function in an anoxic environment, it is not clear how they behave when exposed to air, fed directly into the anode chamber. The current study used cylindrical ceramic MFCs in order to study their behaviour under active aeration conditions. Oxygen in the anode and anolyte flow rate Ceramic MFCs are a cost effective option for scale up [34] and this study looked at their behaviour in a fluctuating environment when air was purposely pumped through the anode chamber alongside the flow of anolyte. The presence of oxygen in the anode chamber could be a problem because dissolved oxygen is found in the treatment process particularly at high flow rates [16] . Key for MFC operation is the transfer of electrons from within the bacterial cell to the anode surface. When oxygen is present in the anode chamber, microorganisms that are metabolically compatible (i.e. aerobic and facultative anaerobic) will oxidise the fuel with the reduction of oxygen rather than using the electrode [25] . While this does not affect the treatment efficiency, it will affect the overall rate of microbial metabolism, power generation and coulombic efficiency. In addition, the presence of oxygen is toxic to strictly anaerobic microorganisms, which in the case of MFCs, could also result in a sub-optimal electrical output. A small number of studies have looked at the influence of oxygen on MFC behaviour [28] and showed that its did not significantly affect power using electro-active monocultures such as the facultative organism Shewanella oneidensis . For this species the presence of oxygen limited the rate of electron transfer to the anode but it also promoted biofilm development, which counteracted the negative impact [22] . Geobacter sulfurreducens is another well-studied electro-active organism and was long thought to be a strict anaerobe, yet it can in fact grow using oxygen as terminal electron acceptor [21] and can produce power in a MFC when oxygen is present providing it is fed from within the anode [25] . A long-term study was performed by De Sá et al [5] who directly exposed areas of the surface of the anode to air. They found a linear relationship between the amount of surface exposed and the degree of inhibition observed by the biofilm. For operation in situ within a wastewater treatment plant, the anodic biofilm will always be a mixed community. Even assuming that the MFC system begins with a single species, over time local organisms in the feedstock are thought to co-colonise or accumulate within the microbial biofilm community, with the whole electrode evolving to suit the prevailing physicochemical conditions. Furthermore, because wastewater is a complex substrate, only a mixed microbial community operating synergistically could deal with the wide mix of organics. Therefore, it is important to know how oxygen might affect such a mixed community. Oh et el. (2009) looked at the effect of oxygen penetrating the proton exchange membrane (PEM) using H-type MFCs being batch-fed [45] . They demonstrated that oxygen did cause the power to drop but that MFCs quickly recovered when the airflow stopped. PEMs can inhibit electro-active bacteria in some conditions [33] and the current study focussed on mixed communities in ceramic MFCs where conditions for electron-abstraction by the anode are more favourable. Moving MFC materials and design away from the conventional H-type and/or cubic type has seen the technology advance from laboratory curiosities towards successful field trials [15] . One of the main factors has been the adoption of ceramic as both the structural material and as medium for ion transfer between the anode and cathode. Initial work looked at pots and cylinders that were configured with the anode inside the vessel and the cathode wrapped around the outside [1] , [2] , [36] . More recently, a commonly used design incorporates the cathode inside the cylinder (sealed at the bottom) and the anode wrapped around the outside [8] . MFCs of this configuration allow the cylinder to be immersed in the anolyte reservoir with the anode exposed to the liquid whilst the cathode sits open to the air. The current study used this design of ceramic cylindrical MFCs and mixed biofilms to explore how they respond to the influx of air directly into the anode chamber. In addition to the introduction of air, the flow rate and hydraulic retention of anolyte is an important consideration. Recently an electrochemical technique for analysing the suitability of materials and operations has been the bi-directional polarisation sweep and this method was incorporated into the study in order to analyse the effects of the flow rate of anolyte on long-term power output performance by the MFCs. Optimising the air-breathing cathode in cylindrical ceramic MFCs The internal cathode design allows liquid catholyte to accumulate, which improves the performance by lowering the ohmic resistance and preventing biofouling [10] . An unresolved engineering challenge with this design has been the poor contact of the cathode with the inner wall of the cylinder. The damp environment can cause the electrode to peel away or become loose as reflected in a drop in power. Another challenge is affixing and selecting the best material for connecting the current collector. Different clays vary in their chemical make-up and so one current-collecting wire might be suitable for one ceramic type but not another. This is because components in the ceramic might be more prone to reacting with the wire materials. In an attempt to address these issues, a unique method was trialled to improve cathode contact by introducing hydrogel powder into the cathode chamber.\n\nIntroducing hydrogel powder in MFC chamber Six large terracotta MFCs ( Table 1 ) were operated for a month with artificial wastewater (See section: MFC construction and operation ) at a flow rate of 5.2 mL per hour before polarisation sweeps were performed. Following polarisation the cathode chambers of three of the MFCs were filled with 8g of hydrogel powder (Stockosorb, Germany). The MFCs were operated for a further 2 months before polarisation sweeps were performed again.",
"discussion": "Results and discussion The effect of air on performance It is inevitable in a continuous flow wastewater treatment environment that oxygen will be present in the liquid. For MFCs positioned in such an environment it is important to understand how they might respond to the presence of oxygen. A number of studies using conventional proton exchange membranes have looked at the effect of oxygen on MFC performance but this is the first time that ceramic cylindrical MFCs have been used to assess the effect, and under conditions that resemble operation at a sequencing batch reactor (SBR). During standard operating conditions, the porosity of the ceramic material will allow partial oxygen to penetrate towards the anode electrode [36] . This will result in a lower open circuit voltage (OCV) because oxygen will contribute to a more positive anode redox potential. However, in a cylindrical configuration when the circuit is closed and electrochemical reactions proceed, in theory less oxygen will infiltrate the anode chamber because it is consumed during the oxygen reduction at the cathode. Interestingly, this could mean that the OCV in many studies using ceramic MFCs is understated and not representative of what might be under closed circuit conditions. This is because, despite ambient conditions being the same, the environment is altered through closed circuit operation. The purpose of the current study however, was the effect of oxygen on closed circuit operation with sequential batch reactor (SBR) operation being the main motivation for the experimental design. Air pumped into the reservoir bottle The first test was to pump air at 4-h intervals into the feed bottle and monitor whether there was any response from the MFCs by the time the feedstock had reached the anode chambers. There was no noticeable effect on electrical output of any of the MFCs and they all performed stably over time (data not shown). This demonstrated that the journey from the reservoir bottle to the MFC was sufficient so that any dissolved oxygen either dissipated or was insufficient to induce an effect. Single injection of air directly into anode chamber In order to observe how the MFCs respond to a single one-off influx of air, using a syringe, 5 mL and then 20 mL of air was injected directly into the MFCs. The lower 5 mL influx of air resulted in negligible change to electrical output (data not shown) however when 20 mL was injected the larger MFCs dropped 8% in power [3% drop in voltage] ( Fig. 2 a and 2b). The smaller fuel cell displayed a more significant drop, with a decrease in power of 42% (19% voltage). Following the drop, the MFCs stabilised before gradually recovering. When the power was normalised to chamber volume the effect is more marked in the smaller MFC, which initially has superior power density, but drops to below the larger MFC ( Fig. 2 c). The greater effect on the smaller MFCs is linked to the proportionately smaller volume of liquid containing dissolved oxygen, making it both quicker to saturate and unsaturate, and a higher proportion of electrode surface exposed to the dissolved air. Furthermore, by proportion, a larger area of chamber was flushed with air which could also negatively impact on the electro-active planktonic organisms. Fig. 3 MFC voltage (under 500 Ω external resistance) in response to 4-h flow of air at different airflow rates through anode chambers; (a) air pumped at 126 mL per hour, (b) 126 mL magnified showing the three MFCs of the triplicate, (c) air pumped at 252 mL per hour, (d) air pumped at 1.1L per hour. Arrows indicate when airflow rate was initiated. Asterisk (*) indicates when anode was flushed with fresh anolyte. [(a), (c) and (d) data = mean and SD (n = 3)]. Fig. 3 Consequently, if the function of the MFCs was to sense the environment as a biosensor, for example to alert to the presence of oxygen, smaller MFCs are preferable because they demonstrated higher sensitivity and an immediate response time. MFCs are a natural biosensor because their output mirrors the microbial responses to the environment. To date, there are a number of reports of MFCs in a sensing role e.g. to monitor biological oxygen demand (BOD) [46] , nitrate [31] , toxic components [47] and other parameters [17] . MFCs have been employed to detect oxygen but these have been using oxygenated liquids fed to the cathode chamber [41] . This type of configuration would not be suitable if the presence of oxygen in the anolyte was of interest. The current study has shown that the response of the anodic biofilm to air could potentially serve a sensing purpose without harming the system. For MFCs that need to be robust against a fluctuating environment, perhaps in the field of wastewater treatment, these data suggest that larger volume MFCs are more desirable. A more thorough examination was carried out next by subjecting the MFCs to longer periods of airflow. 4 h on/4 h off flow of air into anode chambers In order to mimic the sequential aeration conditions inside a SBR, two pumps were alternated every 4 h. Firstly, anolyte was pumped through the anode chambers for 4 h before air was pumped through the anode chamber for another 4 h; this continued in 4-h cycles. Fig. 3 shows the behaviour of three MFCs during the cyclic pumping. For the periods where air was flowing into the chamber (at 126 mL/h) the voltage did drop, but only a few mV (<2%, Fig. 3 a) and the MFCs were not negatively affected by the flow of air through the anode chamber. To highlight this, Fig. 3 b shows a magnified area of the three individual MFCs and the smooth fluctuations during the air followed by feed cycles. The flow of air was then increased to 252 mL/h and initially there was no detrimental impact on MFC performance ( Fig. 3 c). However, after 43 h the MFCs began to decline and with each new influx of air, the decrease became more exaggerated. After approximately 72 h the anode was flushed with fresh anolyte (indicated by asterisk in Fig. 3 c) and the MFCs recovered immediately. When the airflow rate was increased yet further to 1.1L/h, the same pattern was observed where there was some stability before the staggered drop with the first significant decline occurring after 45 h. To test the robustness of the microorganisms, the cycle was allowed to continue for another 25 h before the MFCs were flushed with fresh anolyte and the airflow rate dropped back to 126 mL/h (as indicated by the asterisk in Fig. 3 d). Again the MFCs recovered immediately demonstrating that the constant flow of air did not harm the microbial community responsible for producing power. The reduction in power occurred when the air was flowing and each time the feedstock was introduced the MFCs tried to recover. However, performance did not recover fully to previous levels, because the anolyte flow rate was insufficient to purge the anode chamber of depleted feedstock. Prior to this point the air/oxygen was not inhibiting and the MFCs behaved in a stable manner. However, the air was accelerating the breakdown of organic matter, which is reflected by the decline in electrical output as the feedstock becomes depleted. With each new air introduction the drop becomes more severe because the anolyte has been further depleted, a factor accentuated by the aerobic/facultative organisms present. The COD was measured at two points during the erratic behaviour and COD had dropped by 91% (asterisk in Fig. 3 c) and 96% (asterisk in Fig. 3 d) by the lowest point. When the MFCs were flushed with fresh feedstock the voltage quickly returned to the stable operating output thus demonstrating that the exposure to air had no long-term ill effect on the biofilm. During the 4-h periods when air was flowing, there was no supply of feedstock but the liquid present in the anode chamber during this time was initially nutritious enough to maintain output. However as depletion was accelerated, airflow indirectly became inhibitory to the microbes. When feedstock was reintroduced every 4 h at the flow rate of 5.2 mL/h the electrical output began to pick up but the drops were more severe with each 4-h cycle because effectively a higher proportion of anolyte in the chamber had become exhausted. Furthermore, it is likely that microbes may have started growing inside the feed tube resulting in reduced COD entering the MFC. However, when the anolyte flow rate was temporarily increased (1.1L per hour) in order to replenish the chambers (as indicated by the asterisk) the MFCs quickly recovered. The recovery can be seen in Fig. 4 a, which shows a comparison in the power output of large and small MFCs in identical conditions where the airflow rate was 252 mL/h in 4-h intervals. Interestingly the smaller MFCs took longer (57 h compared to 43 h) for the decline to take effect. This is because the HRT was shorter for the small MFCs (8hrs) than for the large ones (23hrs) and so there was a better recycling retention of fresh feedstock. This is useful to know when considering MFCs for the role of receiving influent from a sequential batch reactor where air is pumped directly. This study demonstrates that the only effect that the cyclic provision of air has on the MFCs is to accelerate the breakdown of COD, which ultimately results in a decline in power. Focussing on the power illustrates how well the MFCs recover after what could be deemed a hostile environment for the bacteria. For example the power density ( Fig. 4 b) drops by 96% in the small and 94% in the large MFCs but recovers fully when fresh feedstock is flushed into the system (at approx. 70 h). The robustness of the organisms is highlighted by the fact that the MFCs have fully recovered within just 2 h. Fig. 4 Comparison between large (120 mL) and small (40 mL) MFCs when subjected to 4 h periods of airflow through anode at 252 mL per hour: (a) power, (b) power density (data: mean and SD, n = 3). Fig. 4 Although no other gases were tested in the current study, there have been other reports to suggest that gas bubbles can improve performance; e.g. bubbles of CO 2 can improve mass transfer through agitation [4] . Furthermore, the presence of oxygen can help MFCs access fuels that normally would not be treated in a strictly anaerobic environment [3] . Effect of anolyte flow rate on bi-directional polarisation When operating MFCs in continuous flow, the flow-rate is vital for maintaining biofilm stability and efficient delivery of nutrients [19] . Too high a flow rate i.e. a lower hydraulic retention time (HRT) will result in an inadequate breakdown of organic matter, and could result in shearing the biofilm, which implies higher maintenance requirements and slow biofilm maturation [29] . A longer HRT will ensure organic matter is more efficiently removed but too long could impact on the biofilm as the organisms receive depleted feedstock. A further complication to operation is the design of the MFC and how the system is configured. In the current study feedstock was fed into the bottom of the anode chamber with the treated effluent leaving via the outlet at the top ( Fig. 1 b). Therefore, organisms colonising the top half of the electrode will have been receiving a more depleted feedstock than those in the lower half of the anode. The nutrient composition particularly received by those at the top of the anode will very much depend on the flow rate. Another factor that flow rate brings is the amount of dissolved oxygen i.e. higher flow rates will deliver more oxygen than the low flow rates [14] . The bi-directional polarisation sweep is a useful tool for assessing the health of the MFC [48] , stability of materials [33] and is important with regards to selecting the right analytical methods [7] . For this study, the technique was used to examine the effect of flow rate on the stability of biofilm over time. At the lower flow rate, which equates to 23 h HRT ( Fig. 5 a), the point of maximum power (MPT) drops by 58% demonstrating that the environment has altered considerably over the course of the first resistance sweep, to the detriment of the microbial community. Over the faster flow rate (HRT 4 h) the MPT still drops between the first sweep and the second but the decline is less severe at 31% ( Fig. 5 b). This demonstrates that at a higher flow rate with more efficient replenishment of nutrient the microorganisms respond positively as epitomised by healthier power curves. In the case of these MFCs, it is hypothesised that higher flow rates may have generated healthier curves with less hysteresis. The bi-directional polarisation sweep could then be a useful tool for determining optimal flow rates based on the extent of hysteresis and this is an area for future work. Fig. 5 The effect of anolyte flow rate on bi-directional power curves generated by 120 mL MFCs at either; (a) 5.2 mL/h or (b) 30 mL/h (data: mean and SEM, n = 3). Fig. 5 Hydrogel in the cathode chamber One of the logistical problems associated with ceramic cylindrical MFCs that incorporate internal cathodes is how to ensure efficient contact between the cathode and the internal wall. Should the cathode peel away or come apart, proton transfer is inhibited and power output diminishes. A number of methods have been adopted to try negating the issue and generally involve holding the cathode material against the wall with a material such as acrylic rings [24] . These methods can cause tearing of the cathode and may inhibit the availability of oxygen at the electrode surface. A new technique was trialled in this study using hydrogel powder. While this is not the first time hydrogel has been used in MFCs, it is the first time it has been employed to improve contact and as a mechanism for collecting catholyte. Hydrogel in its powdered form is used in the cultivation of plants, it retains water in the earth and can hold 150 times its own weight making water more accessible to the roots of plants. Forms of hydrogel have been trialled in MFCs for other roles such as a passive feeding mechanism [37] and as a bridge between electrode and ion exchange membrane to increase cathode potential [49] . In the current study hydrogel powder was poured into the inner cathode chamber after draining the catholyte ( Fig. 6 a). Fig. 6 The cathode chamber of MFCs; (a) before hydrogel powder added, (b) Hydrogel powder added (day 1), (c) after 1 month, and (d) after 2 months. Fig. 6 Prior to the addition of hydrogel, a polarisation sweep was performed on 6 MFCs; 3 which would have hydrogel powder added and three controls that wouldn't. The power curves of all 6 MFCs prior to the addition of hydrogel were comparable (yellow symbols, Fig. 7 a and Fig. 7b) where the average of the hydrogel MFCs was 293 μW (1634 μA) before the addition of hydrogel compared to 300 μW (1642 μA) generated by the MFCs that would remain without hydrogel. Following the polarisation experiment the hydrogel was added to the three hydrogel MFCs and for the next 2 months all MFCs were maintained in identical conditions. During this time the hydrogel became noticeably more swollen as it absorbed any catholyte that was being produced ( Fig. 6 ) and after 2 months had swollen to the brim of the chamber ( Fig. 6 d). Interestingly the conductivity of the hydrogel was higher (15 ± 1 mS/cm) than that of the liquid catholyte (8 ± 1 mS/cm) and the pH was also higher (10.5 ± 0.2) than the liquid catholyte (9.1 ± 0.2). Fig. 7 Power curves showing the effect of adding hydrogel powder into the cathode chamber (a) control MFCs that did not have hydrogel added, before and 2 months later and (b) MFCs that did have hydrogel added, before hydrogel addition and 2 months later (data = mean and SEM, n = 3). Fig. 7 After 2 months, polarisation experiments were performed again on the six MFCs and those without hydrogel addition remained comparable to the output recorded 2 months earlier ( Fig. 7 a). Those MFCs that had the addition of hydrogel displayed significant improvement ( Fig. 7 b) with the maximum power transfer point more than two and a half times higher (825 μW [2775 μA]). This demonstrates that hydrogel can be used to aid performance in future research using ceramic cylinder MFCs by improving the contact between ceramic and cathode. Hydrogel serves the purpose when used in agriculture of reducing the effects of evaporation and leaching by retaining the moisture. In the current study it retained and helped the accumulation of catholyte resulting in a more concentrated sample as reflected by the increased conductivity and pH. Further work will investigate whether the catholyte-hydrogel material might serve a useful purpose such as in an antimicrobial capacity [9] or in the role of atmospheric carbon capture [10] . In addition, further work will investigate the longevity of the hydrogel system and whether it continues to aid performance or whether it will need replacing."
} | 7,240 |
30606121 | PMC6318872 | pmc | 4,137 | {
"abstract": "Background Plants, fungi, and bacteria form complex, mutually-beneficial communities within the soil environment. In return for photosynthetically derived sugars in the form of exudates from plant roots, the microbial symbionts in these rhizosphere communities provide their host plants access to otherwise inaccessible nutrients in soils and help defend the plant against biotic and abiotic stresses. One role that bacteria may play in these communities is that of Mycorrhizal Helper Bacteria (MHB). MHB are bacteria that facilitate the interactions between plant roots and symbiotic mycorrhizal fungi and, while the effects of MHB on the formation of plant-fungal symbiosis and on plant health have been well documented, the specific molecular mechanisms by which MHB drive gene regulation in plant roots leading to these benefits remain largely uncharacterized. Results Here, we investigate the effects of the bacterium Pseudomonas fluorescens SBW25 (SBW25) on aspen root transcriptome using a tripartite laboratory community comprised of Populus tremuloides (aspen) seedlings and the ectomycorrhizal fungus Laccaria bicolor ( Laccaria ). We show that SBW25 has MHB activity and promotes mycorrhization of aspen roots by Laccaria . Using transcriptomic analysis of aspen roots under multiple community compositions, we identify clusters of co-regulated genes associated with mycorrhization, the presence of SBW25, and MHB-associated functions, and we generate a combinatorial logic network that links causal relationships in observed patterns of gene expression in aspen seedling roots in a single Boolean circuit diagram. The predicted regulatory circuit is used to infer regulatory mechanisms associated with MHB activity. Conclusions In our laboratory conditions, SBW25 increases the ability of Laccaria to form ectomycorrhizal interactions with aspen seedling roots through the suppression of aspen root antifungal defense responses. Analysis of transcriptomic data identifies that potential molecular mechanisms in aspen roots that respond to MHB activity are proteins with homology to pollen recognition sensors. Pollen recognition sensors integrate multiple environmental signals to down-regulate pollenization-associated gene clusters, making proteins with homology to this system an excellent fit for a predicted mechanism that integrates information from the rhizosphere to down-regulate antifungal defense response genes in the root. These results provide a deeper understanding of aspen gene regulation in response to MHB and suggest additional, hypothesis-driven biological experiments to validate putative molecular mechanisms of MHB activity in the aspen- Laccaria ectomycorrhizal symbiosis. Electronic supplementary material The online version of this article (10.1186/s12870-018-1610-0) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions A model tripartite association, comprised of aspen seedlings, the ectomycorrhizal fungus L. bicolor , and the PGP P. fluorescens bacterium SBW25, was used to investigate mechanisms of MHB activity through analysis of aspen root transcriptomic data. We demonstrated that SBW25 is a MHB, promoting mycorrhization of aspen seedling roots by Laccaria and that SBW25 persists in the rhizosphere of sand-pot cultures after more than 60 days. We believe this to be the first report of SBW25 possessing MHB activity. A cluster of co-regulated genes in aspen roots was found to strongly correlate with the level of mycorrhization by Laccaria . We propose that global transcription regulation activities in this cluster, including genes for chromatin remodeling and translation activities, indicate a molecular mechanism by which aspen roots change developmental stage from free-living root to mycorrhizal symbiosis. When both Laccaria and SBW25 are present in the rhizosphere community, a co-regulated gene cluster annotated with plant antifungal responses is significantly down-regulated, leading to the hypothesis that MHB-mediated increase in mycorrhization in this system is facilitated through suppression of antifungal defense responses in aspen roots. A putative molecular sensor mechanism for MHB activity in roots was further identified from aspen seedlings. Eight genes with homology to proteins annotated as ‘Recognition of pollen’ are proposed as components of the root’s MHB interactions due to their patterns of expression and their annotation. These proteins can hypothetically act as a biological XOR logic gate that integrates multiple environmental/biotic signals, specifically the simultaneous presence of both Laccaria and SBW25 in the rhizosphere, to down-regulate the aspen root’s fungal-defense response. These model-predicted mechanisms of MHB interactions will inform the design of future biological experiments to validate our proposed molecular mechanisms of MHB activity.",
"discussion": "Discussion Forest trees rely extensively on mycorrhizal symbionts to access limiting soil nutrients in the environment. ECM fungi increase root surface area and contribute new metabolic capabilities to the host tree, all of which alter root-soil interactions. The formation of the ectomycorrhizal symbiosis requires remodeling of the host root immune system, allowing subsequent changes of root structure and function characteristic of fungal-host specific mycorrhizas. This interaction is the result of effectors produced by fungi, including the hormones auxin and ethylene and small secreted effector proteins, that alter host transcription and metabolism to facilitate the colonization of short roots by the ECM fungus [ 20 ]. Changes induced by fungal signals include stimulation of lateral root production as well as changes in cell wall metabolism that foster sites for mycorrhiza formation. In addition to symbiotic mycorrhizal fungi, roots and mycorrhizas are colonized by a diverse bacterial community, many of which similarly alter host physiology and contribute novel metabolic pathways that foster resource acquisition and host environmental stress resistance [ 3 , 25 ]. Further, interactions between microbial symbionts indicate that bacterial-fungal communication influences the ecological structure and function of the rhizosphere community and its interaction with plant roots, with MHB significantly increasing mycorrhization of tree roots [ 11 , 18 , 21 ]. While the molecular signals of some of these interactions have begun to be explored [ 10 , 21 , 26 ], significantly less attention has focused on host molecular response underpinning the tripartite interaction. Pseudomonas fluorescens SBW25 is a mycorrhizal helper bacterium While the presence of SBW25 had a significant, positive effect on short root development and mycorrhization of aspen roots by Laccaria , neither Laccaria nor SBW25, alone or in combination, had a significant effect on aspen seedling shoot or root biomass. The observed increase in mycorrhization cannot, then, be correlated with higher production of photosynthetic compounds from increased leaf biomass or with varying amounts of plant roots exposed to a constant fungal biomass. Therefore, the increase in mycorrhization is due solely to the presence P. fluorescens SBW25 and its impacts on aspen metabolism that enhance symbiosis between aspen and Laccaria . Such an increase in mycorrhization may result from bacteria-induced changes in either host or fungal metabolism and their subsequent interactions (e.g., [ 20 ]) or through more complex interactions that have yet to be elucidated. The formation of ectomycorrhiza requires intricate communication and physical/biochemical interactions that bypass host immune systems designed to protect the root from pathogenic invaders and simultaneously prepare the root for colonization. Hormonal and other effectors produced by fungi stimulate the production of root primordia and alter cell wall biochemistry. The small secreted protein MiSSP7 produced by L. bicolor , for example, has been shown to localize in host root cell nuclei, where host jasmonic acid-responsive gene transcription is suppressed to favor ectomycorrhization [ 26 ]. In addition, the colonizing fungus may regulate the presence/reactivity of its antigenic components, such as chitin, which would reduce host immune response as well [ 20 ]. MHB may intercede in these interactions by predisposing the root for colonization. Kurth et al. (2015) [ 18 ] noted a suite of up-regulated contigs related to perception and signaling in roots of Quercus robur , including members of the salicylic acid and jasmonic acid signaling pathways and significant numbers of Leucine Rich Repeats (LRRs) with homology to LRR-receptor like kinases involved in recognition and signaling. Such changes would be expected to have substantial impact on the receptivity of the root to fungal colonization. MHB may also stimulate mycorrhization by influencing fungal responses fostering colonization. For example, Labbé et al. (2014) [ 11 ] noted Pseudomonas strain-specific gene regulation in L. bicolor related to transcriptional regulatory complexes and biofilm formation, which may play roles in broad fungal metabolic restructuring and fungal-bacterial recognition [ 10 ]. Changes in gene expression associated with signaling pathways and fungal metabolism were also induced by Streptomyces in Amanita muscaria [ 21 ]. In the current study, we evaluated patterns of gene expression in aspen roots in dual and tripartite symbiosis to search for clues underlying the MHB relationship. MHB activity in SBW25 is a function of inhibition of the antifungal defense response in aspen roots The innate immune system is constitutively expressed and is the first line of defense in plants, detecting pathogens as well as other microbes [ 27 ]. When a specific threat is detected by the innate immune system, then expression of genes specific to individual threats are activated and the innate immune system is down-regulated [ 28 ]. The proposed relationship between expression patterns is that the constitutive expression of innate immune response (Cluster 1) when aspen seedlings are cultivated alone is downregulated and exchanged for a more specific antifungal stress response (Cluster 4) when Laccaria is present. In order to confer MHB benefits, the antifungal systems in this cluster would be attenuated when the SBW25 is present. Among the defense responses stimulated by Laccaria are those involved in chitin catabolism as well as numerous ‘response to biotic stress’ genes, which reflect major potential defenses to invading fungal pathogens. When co-inoculated with SBW25, however, these responses are, by-and-large, attenuated and may reflect one of the pathways of MHB action. Among the gene clusters identified, the ‘Global Gene Regulation’ Cluster 2 has the strongest correlation (PCC = 0.99) with observed aspen root mycorrhization and with percent RNAseq reads aligning to SBW25 (PCC = 0.77). Genes in this cluster are up-regulated when Laccaria is present and further up-regulated when Laccaria is co-cultured with SBW25. This cluster is dominated by global transcriptome reprogramming activities as well as mitochondrial function and ATP synthesis genes, which may reflect the outcome of MHB activities: increased colonization increases the demand for energy to support enhanced metabolic demand associated with supporting the mycorrhizal association. Analysis of gene regulatory network as Boolean circuit The evaluation of transcriptomic data above may reflect the outcomes of interactions in the tripartite association leading to increased mycorrhization by MHB, but not necessarily the signals stimulating metabolic changes leading to enhanced colonization. While putative biological functions and the experimental conditions under which they are differentially regulated can be ascribed to co-regulated gene clusters through interpretation of expression patterns (Fig. 3 ) and enriched annotations (Fig. 4 ), we used a Boolean circuit diagram to develop a gene regulatory network that would account for the differentially regulated aspen gene clusters in the tripartite association (Fig. 5 ). Using this approach, Cluster 5 was identified as being the key to this regulatory network. Cluster 5 is controlled by an Exclusive Or (XOR) gate that uses the presence of SBW25 and Laccaria as inputs. An XOR gate is a logical operation that outputs true only when inputs differ, i.e. is activated in this model when either SBW25 or Laccaria is present, but not when both or neither are present. The position of Cluster 5 in the circuit and the preponderance of regulatory-related function annotations noted above (Fig. 4 ) makes this cluster a likely target for aspen root regulatory elements most closely associated with MHB detection and subsequent gene regulation. Aspen membrane-bound sensors with homology to ‘Recognition of Pollen’ proteins are candidate regulators of defense response While the analysis of Boolean logic circuit diagram indicates that the regulation of defense response by Cluster 5 is a key component of MHB activity in this system, the specific molecular mechanisms by which detection of SBW25 by aspen roots regulates aspen root defense response gene expression are not immediately apparent. Assuming that some of the genes responsible for detecting the microbial community are differentially regulated in this experimental system, a set of specific criteria for this sensory mechanism can be hypothesized: (i) present in a cluster regulated by the presence of both Laccaria and SBW25; (ii) enriched in regulatory function annotated as sensors capable of integrating multiple inputs; and (iii) the regulatory gene annotations from (ii) should be uniquely enriched in the cluster identified by criteria (i). For the first criterion, the XOR gate for the presence of SBW25 and Laccaria for activation of Cluster 5 is the best cluster for integrating information about the presence of community members (Fig. 5 ). For the second criterion, considering the sets of enriched annotations (Fig. 4 ), a relevant enriched annotation is ‘Recognition of pollen’ (GO:0048544). This annotation is shared by eight genes in Cluster 5 (Potri.004G028000, Potri.005G014900, Potri.010G103300, Potri.013G121000, Potri.014G086900, Potri.019G119600, Potri.019G119700, and Potri.T021600). For the third criterion, the ‘Recognition of Pollen’ annotation is indeed uniquely enriched in Cluster 5 (Additional file 4 ). Recognition of pollen plays critical roles in controlling plant fertility and involves diverse molecular signatures across species. Recognition is based on small ligands (proteins, glycoproteins, lipids) from pollen that must be recognized by stigmas, with appropriate downstream metabolic responses [ 29 , 30 ]. Receptors also vary extensively, including kinases, RNases, and Ca 2+ -signaling systems [ 30 ]. Given this diversity, and the potential that annotated genes in poplar may be involved in numerous aspects of recognition, the role of proteins with homology to pollen recognition in MHB interactions can be hypothesized. All eight ‘Recognition of pollen’ genes in Cluster 5 are identified as being expressed in root tissue in the DOE Joint Genome Institute database of plant gene data, Phytozome [ 31 ], which suggests both that the detection of expression in these genes in roots in this experiment is not in error and that the actual biological function of these genes in roots is not literally the ‘detection of pollen’. In the stigma, ‘Recognition of pollen’ proteins prevent accidental fertilization by incompatible pollen by integrating two separate signaling molecules present on the pollen grain to down-regulate the genes that initiate pollination [ 32 – 34 ]. This mechanism, i.e. integrating the information from two extracellular signals to down-regulate a suite of related genes, is precisely a match for the biological mechanism of the XOR-gated gene expression patterns in the predicted regulatory network."
} | 4,013 |
37374858 | PMC10303904 | pmc | 4,138 | {
"abstract": "Due to its superior advantages in terms of electronegativity, metallic conductivity, mechanical flexibility, customizable surface chemistry, etc., 2D MXenes for nanogenerators have demonstrated significant progress. In order to push scientific design strategies for the practical application of nanogenerators from the viewpoints of the basic aspect and recent advancements, this systematic review covers the most recent developments of MXenes for nanogenerators in its first section. In the second section, the importance of renewable energy and an introduction to nanogenerators, major classifications, and their working principles are discussed. At the end of this section, various materials used for energy harvesting and frequent combos of MXene with other active materials are described in detail together with the essential framework of nanogenerators. In the third, fourth, and fifth sections, the materials used for nanogenerators, MXene synthesis along with its properties, and MXene nanocomposites with polymeric materials are discussed in detail with the recent progress and challenges for their use in nanogenerator applications. In the sixth section, a thorough discussion of the design strategies and internal improvement mechanisms of MXenes and the composite materials for nanogenerators with 3D printing technologies are presented. Finally, we summarize the key points discussed throughout this review and discuss some thoughts on potential approaches for nanocomposite materials based on MXenes that could be used in nanogenerators for better performance.",
"conclusion": "7. Conclusions and Outlook A comprehensive review has been conducted on composite materials based on MXenes for TENGs and PENGs. The promising potential of nanogenerators is in their ability to harvest mechanical energy, function as multifunctional sensors, and more. The combination of MXenes with PVDF, PDMS, PTFE, PVA, Ag, Au, CNTs, and oxides, benefitting from their superior properties such as electronegativity, metallic conductivity, tunable surface chemistry, and mechanical flexibility, has been shown to positively impact the output performance of nanogenerators. The presence of MXenes in a material can result in a significant increase in electronegativity and conductivity, as well as facilitate fast electron transportation, provide excellent mechanical flexibility, regulate surface charge, and enhance cycling stability and durability. This review may provide guidance for the rational development of advanced nanogenerators that are capable of efficiently harvesting mechanical energy. This field is accompanied by some suggestions and perspectives put forth for consideration. In this field, many works have been completed for energy harvesting applications using polymers and polymeric composite materials. However, real-world problems still exist in analyzing natural systems to harvest green energy. Notably, MXene-based e-skin applications and wearable energy harvesting technologies are very promising areas of work that can be advanced to a higher level. The increasing demand for electronic devices and sensors necessitates the need for MXene-based nanogenerators with higher and more stable output performance to enable smart applications. Further in-depth studies can focus on the synthesis of MXenes, as the techniques used in the synthesis process can significantly impact the intrinsic properties and electrochemical output performance of MXenes. Factors such as flake sizes, defects, and surface chemistry are closely related to the synthesis techniques used. Additionally, several MXenes have only been predicted through theoretical calculations and have yet to be successfully synthesized. The development of additional MXenes and their derivatives can prove to be highly attractive as a source of materials for nanogenerators.",
"introduction": "1. Introduction The sustainable development of people’s lifestyle needs requires emerging green and renewable energy systems, which are the definite solution for global warming and demanding energy requirements [ 1 ]. Moreover, people approach the energy crisis with various personal and social needs, which leads to focus on different kinds of energy harvesting systems [ 2 ]. In the environment, mechanical energy is enormously available in the form of minor and major disturbances. This can be easily converted into useful energy to fulfill current energy requirements. Instead of using these energies, for the most part they are fully wasted or only partially used for energy conversion [ 3 , 4 ]. Nanogenerator types such as piezoelectric and triboelectric are developed to harvest energy from mechanical movements [ 5 , 6 ]. Here, easily accessible and fabricated materials of ceramics, polymers, their composites, and nanocomposites are used for various locations, ambiences, and applications [ 7 ]. Still now, the acquired performance of nanogenerators is comparatively low, which needs to be improved to obtain the highest energy conversion [ 8 ]. There is a big gap between practically gained performances due to the inadequate acquired performance of piezoelectric nanogenerators (PENGs). In addition to piezoelectric nanogenerators, there are also other types of nanogenerators such as triboelectric nanogenerators (TENGs), which generate electrical energy from friction and pyroelectric nanogenerators, which generate electrical energy from temperature changes [ 9 , 10 , 11 ]. Nanogenerators have a number of potential applications in areas such as energy harvesting, self-powered sensors, and wearable electronics [ 12 ]. They can be used to generate electrical energy from ambient vibrations, such as those caused by footsteps or wind, or from human movement such as walking or typing [ 13 , 14 ]. Recent studies have shown that MXenes have high mechanical strength, electrical conductivity, and thermal stability, making them ideal for energy harvesting applications [ 15 , 16 ]. In particular, MXene-based composites have been shown to exhibit improved energy conversion efficiency compared to traditional energy harvesting materials [ 17 ]. For example, MXene-based composites have been shown to be effective for harvesting mechanical energy from ambient vibrations and for generating electricity from temperature gradients [ 18 , 19 ]. In addition, MXenes have been explored for various energy harvesting methods, such as piezoelectric energy harvesting, electromagnetic wave harvesting, and energy storage applications [ 20 , 21 , 22 , 23 ]. Researchers have found that incorporating MXenes into piezoelectric materials can improve the energy conversion efficiency and increase the power output [ 24 ]. The results of previous studies have demonstrated the potential of MXene-based energy harvesting for a variety of energy sources [ 25 ]. Further research and development in this field is expected to lead to even more efficient and effective energy harvesting technologies in the future [ 26 , 27 ]. The voltage output of MXene-based nanogenerators depends on several factors, including the specific MXene material used, the configuration of the device, and the mechanical energy input [ 28 ]. In general, MXene-based nanogenerators have demonstrated high voltage output, with some studies reporting voltage outputs of up to several volts under optimal conditions [ 29 ]. The high voltage output of MXene piezoelectric nanogenerators is due to the strong piezoelectric properties of MXene materials, which allow for efficient conversion of mechanical energy into electrical energy [ 24 , 30 ]. Additionally, the voltage output of MXene piezoelectric nanogenerators can be improved by optimizing the device configuration and by using MXene materials with higher piezoelectric coefficients [ 31 , 32 ]. It should be noted that the voltage output of MXene piezoelectric nanogenerators is typically small and is not enough to power most electronic devices on its own [ 33 ]. However, the small voltage output can be used to power low-power devices, such as sensors, or can be stored in a battery for later use [ 34 ]. MXene piezoelectric nanogenerators have demonstrated promising voltage output and have the potential to be used for applicable energy harvesting applications. There is ongoing research to further optimize the performance and stability of these devices for practical applications [ 18 , 35 , 36 ]. This paper presents the latest developments in MXene-based materials, especially for triboelectric and piezoelectric nanogenerators. Figure 1 shows the overview of MXene-based materials for energy harvesting. To begin, this review covers the fundamental background of piezoelectric and triboelectric nanogenerators, encompassing their key parameters, working mechanisms, and challenges. Furthermore, this review highlights the superior properties of MXene-based materials for piezoelectric and triboelectric nanogenerators. Subsequently, this review comprehensively discusses the design tactics and mechanisms of MXenes in enhancing the output performance of nanogenerators. Finally, this review offers some perspectives and suggestions on future design tactics for MXene-based composite materials in the development of advanced nanogenerators."
} | 2,303 |
28579915 | PMC5434129 | pmc | 4,145 | {
"abstract": "Mesophotic coral ecosystems (MCEs) are generally poorly studied, and our knowledge of lower MCEs (below 60 m depth) is largely limited to visual surveys. Here, we provide a first detailed assessment of the prokaryotic community associated with scleractinian corals over a depth gradient to the lower mesophotic realm (15–85 m). Specimens of three Caribbean coral species exhibiting differences in their depth distribution ranges ( Agaricia grahamae , Madracis pharensis and Stephanocoenia intersepta ) were collected with a manned submersible on the island of Curaçao, and their prokaryotic communities assessed using 16S rRNA gene sequencing analysis. Corals with narrower depth distribution ranges (depth-specialists) were associated with a stable prokaryotic community, whereas corals with a broader niche range (depth-generalists) revealed a higher variability in their prokaryotic community. The observed depth effects match previously described patterns in Symbiodinium depth zonation. This highlights the contribution of structured microbial communities over depth to the coral’s ability to colonize a broader depth range. Electronic supplementary material The online version of this article (doi:10.1007/s00338-016-1517-x) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction Mesophotic coral ecosystems (MCEs) represent an extension of shallow-water coral reefs and provide an extensive habitat for light-dependent corals in subtropical/tropical regions starting at 30–40 m and reaching down to depths of about 150 m (Lesser et al. 2009 , Hinderstein et al. 2010 ). MCEs can be further divided into the “upper mesophotic” (30–60 m) and “lower mesophotic” (>60 m), with the first representing a transition zone between shallow-water and lower mesophotic communities (sharing species of opposing depth zones) and the latter representing a more specialized coral community (Bongaerts et al. 2010 , 2015b ; Kahng et al. 2010 ). Given the increased difficulty in accessing lower mesophotic depths, most studies have thus far been limited to visual surveys of the benthos, and consequently, little is known about the coral-associated microbial communities. A recent molecular study demonstrated that lower mesophotic depths in the southern Caribbean harbor a genetically distinct coral and associated Symbiodinium community, likely reflecting specialization of both symbiotic partners to the mesophotic environment (Bongaerts et al. 2015b ). The coral holobiont, however, harbors not only symbiotic phototrophic zooxanthellae but also numerous other microorganisms such as other protists, fungi, bacteria, archaea and viruses (Rohwer et al. 2002 ; Carlos et al. 2013 ). Prokaryotes, including bacteria and archaea, are of particular interest due to their diverse metabolic and functional capabilities within the coral holobiont and their potential to complement the metabolic needs of the coral host (Ainsworth et al. 2015 ; Thompson et al. 2015 ). Studies on shallow-water communities have shown that prokaryotic communities associated with corals are species specific (Rohwer et al. 2002 ) and have a significant influence on host resilience (Glasl et al. 2016 ). Another study has found coral microbiomes in Seriatopora hystrix to correlate to reef habitat (depth) and geographical location, but not to intrinsic factors such as host genetic lineage and Symbiodinium genotype (Pantos et al. 2015 ). Although the interest in coral–prokaryote interactions has increased over the last decade (reviewed by Thompson et al. 2015 ), only sparse information on the prokaryotic community associated with corals from the upper mesophotic is available (reviewed by Olson and Kellogg 2010 ) and data from lower MCEs are virtually non-existent (but see Ainsworth et al. 2015 ), despite the potential metabolic contribution of these communities (compared to Symbiodinium ) to the energetic balance of corals given the extremely low light conditions at lower mesophotic depths (reviewed by Thompson et al. 2015 ). Here, we provide a first assessment of the variation in the structure and composition of coral-tissue-dwelling prokaryotic communities from shallow reef habitats down to lower mesophotic depths in three common Caribbean coral species with broad, but distinct, depth distributions on the island of Curaçao. In this study, we aimed at (1) determining whether the lower mesophotic corals host a distinct prokaryotic community or indicator assemblages and (2) addressing the respective roles of depth and host in prokaryotic community structure.",
"discussion": "Results and discussion Prokaryotic communities associated with the tissue of the corals were structured according to coral species and depth (permutation test CCA; p < 0.001 and p < 0.01, respectively; Electronic Supplementary Material, ESM, Table S1). As sampling location did not influence prokaryotic community composition, samples from both locations (ESM Table S2) were merged for further analysis. There were no significant differences in alpha diversity, richness or evenness of the prokaryotic communities (based on prokaryotic families) among the three studied coral species or in response to depth (within each species; ESM Tables S3, S4, S5). Consequently, the community composition, rather than alpha diversity, is responsible for the observed variation in prokaryotic communities across sampling depths and among host species. Tissue-associated prokaryotic community composition differed significantly (PERMANOVA, p < 0.05; Tables S6, S7) among A. grahamae , M. pharensis and S. intersepta at a single depth (55 m), which seems driven primarily by the large differences between A. grahamae and M. pharensis (Fig. 1 a). Similar results (not shown) were obtained when the analysis was carried out with presence/absence data. These results are consistent with the widespread host specificity of prokaryotic community composition over space and time (Rohwer et al. 2002 ). Fig. 1 Non-metric multidimensional scaling ordination visualizing the prokaryotic community structure based on relative abundance of prokaryotic families ( a ) among the three different host species for a single depth (at 55 m) ( b, c, d ) within Agaricia grahamae, Madracis pharensis and Stephanocoenia intersepta, respectively, over their natural depth range (at 15, 55 and 85 m) \n There were no significant differences in the prokaryotic community assemblage of A. grahamae , a deep-water specialist (Fig. 1 b), between upper and lower depth populations (55 vs. 85 m; ESM Tables S8, S9). In contrast, S. intersepta , a depth-generalist (Fig. 1 c), showed significant difference between depth zones, but only with presence/absence data (PERMANOVA, p < 0.01; ESM Tables S10, S11). This suggests that the rare or low-abundance prokaryotic families are driving the differences between depths within this species. Finally, M. pharensis, an “extreme” depth-generalist (Fig. 1 d), showed significant depth variation (PERMANOVA, p < 0.01; ESM Tables S12, S13). The shallow (15 m) and the deep (85 m) populations of M. pharensis significantly varied in their prokaryotic community composition (PERMANOVA, p < ,0.01; ESM Table S14). Madracis pharensis samples from 55 m, however, seem to overlap with communities originating from the depth extremes and may therefore represent a transition from shallow water to the lower mesophotic reef. Similar results were obtained with presence/absence data (except for S. intersepta ). Overall these results suggest that “depth-specialist” hosts, characterized by their restricted depth distribution (Bak 1977 ; Bongaerts et al. 2010 ), maintain a specific holobiont community. “Depth-generalist” hosts with a wide depth distribution (Bak 1977 ; Bongaerts et al. 2010 ), however, might host the most favorable prokaryotic composition for the surrounding environment (as also shown by Pantos et al. 2015 ). We hypothesize that this association of corals with a range of different prokaryotes over depth greatly contributes to host distribution and survival across the different depth habitats. Overall no single prokaryotic family was identified as universal depth indicator across all studied coral species (Fig. 2 ). Thus, the prokaryotic community seems to be generally shaped by its host rather than by predominant external environmental parameters (here represented by depth). However, depth-indicator prokaryotic taxa were identified within each individual coral species. Agaricia grahamae and S. intersepta hosted in total two and six prokaryotic taxa, respectively, that were identified as depth indicators (Fig. 2 ). In contrast to the species with more restricted depth distributions, M. pharensis harbored 14 prokaryotic taxa that were significantly associated with at least one particular depth zone (Fig. 2 ). For example, bacteria of the order Chloroflexales were significantly (IndVal; p < 0.05) associated with M. pharensis at 15 m depth, showed a steep decrease in their relative abundance toward the upper mesophotic and were totally absent in the lower mesophotic. The bacterial family Amoebophilaceae showed the opposite trend, with an increase in relative abundance with depth within M. pharensis , and were identified as a significant indicator (IndVal; p < 0.05) at 55 and 85 m depth. This is the first time that this bacterial family of known obligate intracellular amoeba symbionts (Schmitz-Esser et al. 2010 ) is recognized as an ecologically relevant member of the coral intratissue microbiome, an observation warranting further investigation. Overall, there is also a tendency for a higher relative abundance of cyanobacteria with increasing depth. This increase could relate to a modulation of Symbiodinium vs Cyanobacteria populations in holobionts reliant on photosynthetic input for nutrition (Lesser et al. 2007 ). Although further prokaryotic indicator taxa can be seen in Fig. 2 , their exact function and metabolic potential remain elusive and require further investigation. Fig. 2 Relative abundance of indicator prokaryotic taxa associated with Agaricia grahamae ( A.g. ) , Madracis pharensis ( M.p. ) and Stephanocoenia intersepta ( S.i. ) at 55 m and among sampling depths (15, 55 and 85 m) for each individual coral species. Indicator taxa were identified with indicator values analysis to be significantly ( p < 0.05) associated with a certain sampling host or depth group (indicated by colored circles ) \n The observed spatial dynamics in prokaryotic community composition over depth for these three studied coral species closely resemble those reported earlier for their dominant Symbiodinium communities (as determined within the detection limits of ITS2-DGGE by Bongaerts et al. 2015a ). Agaricia grahamae , for example, neither exhibits zonation in the genetic lineages of its associated Symbiodinium (type C3/C11 is ubiquitous, Bongaerts et al. 2015a ) nor in the prokaryotic community over depth. In contrast, M. pharensis shows both a significant shift in the Symbiodinium community (type B7 in the shallow layers is completely replaced by type B15 in the mesophotic habitats; Bongaerts et al. 2015a ) and in its prokaryotic community composition. Finally, S. intersepta exhibits a shift in the Symbiodinium community over its depth range (mixed communities changing from C16 to C3 and C1 as dominant types with increasing depth; Bongaerts et al. 2015a ), as well as a shift in its prokaryotic community (based on presence/absence data). Although we cannot decisively differentiate between the effect of depth and the effects of the holobiont itself, there is evidence that the holobiont ( Symbiodinium and/or coral host) modulates the prokaryotic community associated with the coral tissue (Ainsworth et al. 2015 ). This conclusion is in contrast with that of Pantos et al. ( 2015 ) for Seriatopora hystrix ; they suggested that the variation in microbial communities associated with coral hosts is primarily driven by external environmental conditions. However, our study focused on the detection of prokaryotes associated with relatively stable intratissue microenvironment, whereas Pantos et al. ( 2015 ) likely included a large portion of coral surface mucus, whose associated prokaryotic communities are more exposed to the ambient reef environment and, therefore, more likely to vary spatially. This study provides the first detailed assessment of the prokaryotic community associated with multiple scleractinian corals toward the lower mesophotic reef. The depth distribution range of coral species seemed to affect the overall variability of the prokaryotic community associated with coral tissue. Coral species with narrower depth distribution ranges retained a stable prokaryotic community, whereas corals with a broader depth distribution revealed higher taxonomic flexibility in their associated prokaryotic community. The observed depth effects are consistent with earlier published Symbiodinium variation (Bongaerts et al. 2015a ). This highlights the contribution of structured microbial communities over depth to the coral’s ability to colonize a broader depth range."
} | 3,312 |
37894861 | PMC10607142 | pmc | 4,146 | {
"abstract": "Various kinds of plastics have been developed over the past century, vastly improving the quality of life. However, the indiscriminate production and irresponsible management of plastics have led to the accumulation of plastic waste, emerging as a pressing environmental concern. To establish a clean and sustainable plastic economy, plastic recycling becomes imperative to mitigate resource depletion and replace non-eco-friendly processes, such as incineration. Although chemical and mechanical recycling technologies exist, the prevalence of composite plastics in product manufacturing complicates recycling efforts. In recent years, the biodegradation of plastics using enzymes and microorganisms has been reported, opening a new possibility for biotechnological plastic degradation and bio-upcycling. This review provides an overview of microbial strains capable of degrading various plastics, highlighting key enzymes and their role. In addition, recent advances in plastic waste valorization technology based on systems metabolic engineering are explored in detail. Finally, future perspectives on systems metabolic engineering strategies to develop a circular plastic bioeconomy are discussed.",
"conclusion": "5. Conclusions Although the use of plastics is widespread and cost-effective, the prevailing linear plastic lifecycle significantly contributes to environmental pollution and resource depletion in our current plastic era. This review delves into the realm of plastic-degrading enzymes and their associated microorganisms. While significant research efforts have been devoted to PET degradation, with PETase undergoing extensive engineering, other major plastics such as PE, PP, PVC, PU, and PS lack specific degrading enzymes identified thus far. In addition, this review explores the value-added products obtained from bio-upcycling degraded plastic products, considering the framework of systems metabolic engineering. While the substantial challenges that need to be addressed to achieve industrial-level efficiency are acknowledged, the rapid advancement of available tools in this field underscores the expectation that the incorporation of systems metabolic engineering in plastic degradation and bio-upcycling will offer a fundamental solution for addressing the treatment of plastic waste.",
"introduction": "1. Introduction Plastics have become an irreplaceable material in our daily lives due to their desirable properties like corrosion resistance, durability, ease of fabrication, lightweight nature, cost-effectiveness, and transparency. Driven by high consumer demand, global plastic production reached 460 million tons in 2019 and is projected to rapidly increase to reach 1 billion tons by 2050 [ 1 ]. Due to over 150 years of effort in improving the properties of plastics, most plastics are highly resistant to natural degradation (e.g., bio-, photo-, and thermo-oxidation), requiring centuries for complete degradation [ 2 ]. In addition, only 9% of post-consumer plastics undergo recycling, while the majority end up either incinerated (12%) or disposed of in landfills (79%), due to weak plastic waste regulation/supervision, a lack of requisite infrastructure, and the collection/sorting cost of plastic waste [ 1 ]. As a result, over 4.8 billion tons of plastic waste have accumulated in poorly managed landfills, causing significant disruptions to ecosystems and directly impacting human health and social well-being [ 3 ]. Thus, it is unquestionable that environmental pollution from the accumulation of plastic waste has evolved into a grave global concern. To address the growing issue of plastic waste accumulation, several recycling methods have been developed. Mechanical recycling, a well-established technique involving the grinding or pelletizing of plastics into small particles for reuse in new products, is perhaps the most mature technology, owing to its simplicity and intuitive approach to recycling [ 4 ]. However, post-consumer plastic waste composed of mixed plastics, non-plastic materials, and additives requires sorting and cleaning before mechanical recycling, and failure to do so often leads to down-cycling into low-quality products. Moreover, the number of times plastics can be mechanically recycled is limited as the recycled plastic deteriorates through plastic grinding or pelletizing [ 5 ]. Chemical recycling processes such as gasification, pyrolysis, and chemolysis have the capacity to break down plastics into oligomers, monomers, and gaseous products, making them suitable for handling post-consumer plastic waste [ 6 ]. However, these processes are frequently energy-intensive, utilize large quantities of chemicals, and produce greenhouse gases and toxic residues. It is expected that approximately 2.8 gigatons of CO 2 per year will be emitted into the atmosphere by 2050 from plastic incineration [ 1 ]. Therefore, ongoing efforts are aimed at exploring alternative technologies to establish an efficient and environmentally responsible plastic waste management system, fostering a sustainable plastic economy. Microbial or enzymatic degradation of plastic waste offers a compelling alternative for plastic waste management, driven by several key advantages: (1) microorganisms have continuously evolved their metabolic capacity by creating new enzymes and extending metabolic pathways to incorporate different anthropogenic compounds (e.g., plastic) into their cellular system; (2) microorganisms and enzymes function under mild temperature and pressure conditions, without the need for toxic chemicals; (3) degradation of post-consumer plastic waste is possible without the necessity of prior sorting or cleaning. In addition, the advancements in systems metabolic engineering have accelerated the development of microbial cell factories capable of high-performance production of chemicals, fuels, and materials [ 7 ]. Hence, the degradation of plastic waste and bio-upcycling into value-added bioproducts can be integrated into a single bioprocess, which sets this approach apart from other plastic waste management technologies. This capability for biodegradation of plastic waste holds great promise in establishing a cleaner and more sustainable plastic economy ( Figure 1 ). The review provides a comprehensive summary of the current advances in microbial strains capable of degrading various plastics as well as key enzymes and their role in plastic degradation. Moreover, detailed information on the development of plastic waste valorization technology based on systems metabolic engineering is provided. Finally, future perspectives on establishing a circular plastic bioeconomy are discussed."
} | 1,661 |
40143859 | PMC11937098 | pmc | 4,147 | {
"abstract": "Introduction Soil microbiome transplantation is a promising technique for enhancing plant holobiont response to abiotic and biotic stresses. However, the rapid assessment of microbiome-plant functional integration in short-term experiments remains a challenge. Methods This study investigates the potential of three evergreen sclerophyll species, Pistacia lentiscus (PL), Rosmarinus officinalis (RO), and Juniperus phoenicea (JP), to serve as a reservoir for microbial communities able to confer enhanced tolerance to drought in Salvia officinalis cultivated under water shortage, by analyzing biomass production, plant phenotype, plant ecophysiological responses, and leaf metabolome. Results Our results showed that the inoculation with the three rhizomicrobiomes did not enhance total plant biomass, while it significantly influenced plant architecture, ecophysiology, and metabolic responses. The inoculation with the JP rhizomicrobiome led to a significant increase in root biomass, resulting in smaller leaves and a higher leaf number. These morphological changes suggest improved water acquisition and thermoregulation strategies. Furthermore, distinct stomatal conductance patterns were observed in plants inoculated with microbiomes from PJ and PL, indicating altered responses to drought stress. The metabolome analysis demonstrated that rhizomicrobiome transplantation significantly influenced the leaf metabolome of S. officinalis . All three rhizomicrobiomes promoted the accumulation of phenolic compounds, terpenoids, and alkaloids, known to play crucial roles in plant defense and stress response. Five molecules (genkwanin, beta-ionone, sumatrol, beta-peltatin-A-methyl ester, and cinnamoyl-beta-D-glucoside) were commonly accumulated in leaves of inoculated sage, independently of the microbiome. Furthermore, unique metabolic alterations were observed depending on the specific inoculated rhizomicrobiome, highlighting the specialized nature of plant-microbe interactions and the possible use of these specific molecules as biomarkers to monitor the recruitment of beneficial microorganisms. Discussion This study provides compelling evidence that microbiome transplantation can induce phenotypic and metabolic changes in recipient plants, potentially enhancing their resilience to water scarcity. Our findings emphasize the importance of considering multiple factors, including biomass, physiology, and metabolomics, when evaluating the effectiveness of microbiome engineering for improving plant stress tolerance.",
"conclusion": "5 Conclusion This study demonstrates that inoculation with microorganisms can induce significant changes in plant morphology, physiology, and resource allocation, significantly influencing plant responses to drought. Notably, inoculation with the rhizomicrobiome from J. phoenicea led to increased root biomass, potentially enhancing water and nutrient uptake. This treatment also induced a reduction in leaf size, which may improve thermoregulation, reducing oxidative stress. Furthermore, inoculation with rhizomicrobiomes from J. phoenicea and P. lentiscus resulted in distinct stomatal conductance patterns, suggesting altered water-use strategies. Metabolomic analysis revealed that microbiome transplantation induced substantial reprogramming of the leaf metabolome. Inoculation with all three rhizomicrobiomes led to the accumulation of secondary metabolites associated with stress tolerance, including flavonoids, phenylpropanoids, and apocarotenoids. Specifically, the rhizomicrobiome from J. phoenicea triggered a more balanced metabolic response, with moderate upregulation of a diverse range of metabolites. These results highlight the importance of using a multidisciplinary approach to evaluate the efficacy of selected transplanted microbial communities in enhancing plant stress tolerance, especially in slow-growing species in which differences in biomass production in the short term might not occur. This approach holds promise for the selection and application of microbiomes in revegetation programs and sustainable agriculture in semiarid Mediterranean ecosystems facing increasing water scarcity due to climate change. Further research is needed to fully elucidate the relation between plant responses and the microbiome composition, understand the mechanisms involved and explore the potential applications of these findings in sustainable agriculture and ecosystem management.",
"introduction": "1 Introduction The plant-associated microbiome, a complex consortium of microorganisms inhabiting the phyllosphere, rhizosphere, and endosphere, constitutes a vast reservoir of genetic diversity that plays a crucial role in plant health and fitness ( Trivedi et al., 2020 ; Bhattacharjee et al., 2022 ). This intricate microbial network provides many benefits to the host plant, including enhanced nutrient acquisition and suppression of phytopathogens, ultimately contributing to increased plant productivity and survival ( Trivedi et al., 2020 ; Bhattacharjee et al., 2022 ). The composition and function of the plant microbiome are dynamically shaped by a complex interplay of factors, including host genotype, soil properties, developmental stage, microbial competition, and environmental stressors ( Pérez-Izquierdo et al., 2019 ; Trivedi et al., 2020 ; Mahmud et al., 2021 ; Berruto and Demirer, 2024 ). Consequently, elucidating the mechanisms by which these microbial communities, mainly bacteria and fungi, confer beneficial effects on plant growth and development has become a major focus of research ( Abou Jaoudé et al., 2023 ). Plant growth-promoting rhizobacteria (PGPR) are known for their ability to enhance the nutritional status of plants through diverse mechanisms. These mechanisms include direct nutrient provision ( Fürnkranz et al., 2008 ; Moreau et al., 2019 ), the conversion of recalcitrant nutrients into forms that are available to plants ( Lorenzi et al., 2022 ; Raymond et al., 2021 ; Kumawat et al., 2021 ; de Andrade et al., 2023 ), and the enhancement of nutrient uptake efficiency. The latter is achieved through increased root length ( Mantelin et al., 2006 ; Apine and Jadhav, 2011 ; Ferreira Rêgo et al., 2014 ; Marín et al., 2021 ) and enhanced lateral root development ( Mantelin et al., 2006 ; Vanegas and Uribe-Vélez, 2014 ; Azizi et al., 2022 ). Consequently, integrating microbial biotechnology, particularly by harnessing beneficial plant-microbe interactions, presents a promising strategy for optimizing plant fitness and productivity and mitigating future food security challenges. Exploiting the plant microbiome’s potential has driven the emergence of microbiome engineering, a field focused on utilizing microorganisms for enhancing plant growth ( Berruto and Demirer, 2024 ). This approach encompasses various strategies, including the application of single or consortia of probiotic microbial strains possessing specific growth-promoting traits, the use of host plants to recruit beneficial microbiome members selectively, the modification of soil properties to stimulate the growth and activity of desirable microorganisms and microbiome transplantation ( Song et al., 2021 ). Microbiome transplantation involves transferring a microbial community, along with its associated functional capabilities, from a donor to a recipient host. This method has been shown to be a more comprehensive and resilient approach to promoting plant growth compared to single-strain inoculations ( Jousset and Lee, 2023 ). This technique has emerged as a promising strategy to overcome the limitations associated with single-strain or consortia inoculations, which often face challenges regarding survival and efficacy within complex environmental settings ( Ray et al., 2020 ; Park et al., 2023 ). Manipulation of the plant microbiome via transplantation can be achieved through various methods, including a “wash procedure” involving the inoculation of concentrated microbial communities obtained from healthy donor plants via centrifugation ( Toju et al., 2018 ) or through direct transfer of soil from the donor plant’s rhizosphere ( Howard et al., 2017 ). This manipulation ideally occurs during the initial stages of plant development to maximize the influence of the introduced microbiome on the recipient plant’s microbial community. However, microbiome transplantation has often proven to be a trial-and-error process with a high failure rate ( Choi et al., 2020 ). This lack of consistent success is likely attributed to unpredictable interactions and coalescence processes between the transplanted microbiome and the recipient plant’s existing microbial community ( Rillig et al., 2016 ), as well as potential incompatibilities between the introduced microbiome and the host plant itself ( Jousset and Lee, 2023 ). These challenges underscore the need for further research to understand the complex dynamics of microbiome transplantation and improve its efficacy. While microbiome transplantation offers significant potential, its success depends on carefully selecting and screening donor microbiomes. Current evaluation methods primarily rely on analyzing plant responses, such as disease suppression or biomass increase, often coupled with assessing changes in soil quality and microbiome composition. For example, Wei et al. (2019) , in a study aiming at exploring how initial soil microbiome composition influences disease response in tomatoes, found that the presence of rare specific taxa, such as pathogen-suppressing Pseudomonas and Bacillus and high abundance of genes encoding non-ribosomal peptide and polyketide synthases (antimicrobial compounds) predict plant survival to the plant pathogenic Ralstonia solanacearum bacterium. The next tomato generation planted in these soils was analyzed for disease incidence. A decrease in visible symptoms demonstrated that microbiome-mediated plant protection could be transferred via soil transplantation. Jiang et al. (2022) found that the rhizosphere microbiome of resistant varieties was enriched for distinct and specific bacterial taxa associated with disease suppression. The microbiome transplant efficacy was quantified using source tracking analysis, i.e., DNA-based techniques to identify the specific types of microorganisms present in the rhizosphere of recipient plants. While the presence of specific taxa can provide valuable insights into the potential benefits of a microbiome, relying solely on plant biomass assessment and microbial composition analysis may provide an incomplete understanding of the intricate interplay between the transplanted microbiome and the host plant. Therefore, it is essential to integrate compositional analysis with functional studies that thoroughly investigate the dynamic interactions between the microbiome and the host to elucidate the functional mechanisms underlying microbiome-mediated plant growth promotion. This approach should examine gene expression, metabolome profiling, and physiological responses in both the plant and the microbiome, particularly during the early stages of development, which can be challenging to assess in slow-growing species. Furthermore, while microbiome composition is crucial for evaluating its safety, a comprehensive understanding of its functional integration with the host plant is paramount for harnessing its full potential to enhance plant growth, development, and stress resilience. Drought is a primary cause of crop yield reduction, posing a significant threat to global food security ( Reddy et al., 2004 ; Gupta et al., 2020 ). Plants have evolved various mechanisms to cope with water scarcity, including (1) modifications in root architecture to enhance water uptake, (2) stomatal closure regulated by hormonal signals to minimize water loss, (3) the accumulation of metabolites to adjust osmotic pressure, (4) the dissipation of excess energy and the synthesis of metabolites to mitigate oxidative stress arising from the accumulation of reactive oxygen species (ROS) due to an impaired consumption between energy production (NADPH-H + and ATP) in the light reaction and consumption in the Calvin cycle ( Selmar and Kleinwächter, 2013 ; Caser et al., 2019 ; Kollist et al., 2019 ; Gupta et al., 2020 ). Consequently, drought causes a reprogramming of plant metabolism, affecting enzyme activity, substrate availability, and the demand for specific stress-responsive primary (sugars, polyols, amino acids) and secondary metabolites ( Kumar et al., 2021 ). Salvia officinalis L. (sage) is a valuable medicinal and aromatic shrub native to the Mediterranean region, an environment characterized by prolonged periods of water deficit and high temperatures ( Savi et al., 2016 ; Valkovszki et al., 2023 ). This species is renowned for its essential oil, rich in terpenoids and phenolics, contributing to its numerous biological activities ( Grdiša et al., 2015 ). While generally tolerant to water scarcity, severe drought can negatively impact Salvia species ( Caser et al., 2019 ; Khodadadi et al., 2023 ; Li et al., 2023 ). This study explores the potential of leveraging specialized rhizosphere microbiomes from stress-tolerant Mediterranean aromatic plants ( Rosmarinus officinalis L., Pistacia lentiscus L., and Juniperus phoenicea L.) to enhance the resilience and productivity of Salvia officinalis L. (sage) under water-limited conditions. Plants thriving in marginal environments often harbor unique microbial communities with enhanced adaptive capabilities shaped by co-evolutionary processes under challenging conditions ( Meena et al., 2017 ). These specialized microbiomes represent an untapped resource for discovering novel microbial biostimulants with the potential to overcome the limitations of existing products and contribute to sustainable agricultural practices. Furthermore, the rhizomicrobiome of medicinal plants in such environments exhibits rich microbial diversity, driven by the selective pressure exerted by root exudates and secondary metabolites ( Jabborova et al., 2024 ), increasing the likelihood of harboring rare taxa with beneficial traits. This study employs a multidisciplinary approach to (1) evaluate the ability of the transplanted microbial communities to enhance drought resistance in sage and (2) elucidate the functional integration of these microbiomes with the host plant by investigating the architectural, ecophysiological, and metabolic responses of S. officinalis under severe drought conditions. The goal is to develop effective tools for early identification of beneficial microbiomes or specific strains, enabling their selection before the full microbiome composition is defined, particularly in slow growing species subjected to stress.",
"discussion": "4 Discussion Microbiome engineering, achieved through the transplantation of microbial communities from donor to recipient plants, is a promising biotechnology with the potential to enhance plant traits and survival under biotic or abiotic stress. Panke-Buisse et al. (2015) utilized Arabidopsis thaliana Col. in a multi-generational experimental system to select soil microbiomes that induced earlier or later flowering times in their hosts. They demonstrated that distinct microbiota profiles were assembled by flowering time treatment, and subsequent inoculation with these microbial communities induced flowering time modifications in both A. thaliana and Brassica rapa . Moreover, microbiome transplantation has shown considerable potential in mitigating plant diseases ( Kwak et al., 2018 ; Wei et al., 2019 ; Bziuk et al., 2022 ; Jiang et al., 2022 ; Khatri et al., 2024 ), facilitating plant growth in contaminated soils ( Yergeau et al., 2015 ), and enhancing plant resilience to abiotic stress factors ( Zolla et al., 2013 ). Beyond the scope of agricultural applications, microbiome-based interventions are gaining traction in farming settings and natural ecosystems. These interventions offer a promising strategy for restoring biodiversity and enhancing the resilience of wildlife and ecosystems. Notably, microbiome transplantation has been shown to improve tree growth and survival under drought and heat stress when recipient trees are inoculated with microbial communities from harsh environments ( Allsup et al., 2023 ). This approach holds significant potential for mitigating climate change impacts, including projected reductions in winter rainfall of 15% by 2030 and 30% by 2070 ( IPCC, 2022 ). In this study, we evaluated the potential of three evergreen sclerophyll species, P. lentiscus , R. officinalis, and J. phoenicea , seasonally subjected to drought, to serve as a reservoir for microbial communities able to confer enhanced drought resistance traits to S. officinalis cultivated under water shortage, by analyzing biomass production, plant phenotype, and leaf metabolome. S. officinalis is a typical species inhabiting the Mediterranean Basin characterized by semi-arid soils, long-term decrease in water availability, and extremely high air temperatures and irradiance ( Armada et al., 2013 ; Savi et al., 2016 ). Despite its tolerance to drought, S. officinalis is adversely affected by prolonged reductions in soil water potential. Grisafi et al. (2017) observed decreased stomatal conductance, net photosynthesis, and leaf area in S. officinalis under drought conditions. Savi et al. (2016) further demonstrated that the leaves of Salvia spp. exhibited a decline in water transport efficiency at water potential values that are more typical of mesophyte species than of xerophyte species. These observations highlight the complexity of drought responses in sage and suggest that introducing beneficial rhizomicrobiomes may offer a strategy to enhance its resilience to water scarcity, potentially mitigating the negative impacts of drought on physiological processes and growth. S. officinalis is a valuable species for revegetation programs in semiarid Mediterranean ecosystems. Enhancing plant establishment by directly applying bacterial inocula may benefit these efforts ( Armada et al., 2013 ). The composition of a microbiome is significantly influenced by the host plant ( Santoyo, 2022 ), plant–plant interactions ( Abou Jaoudé et al., 2024 ; Newberger et al., 2023 ), and environmental growth conditions ( Postiglione et al., 2022 ). Idbella et al. (2022) identified differences in the composition of the rhizomicrobiomes of several Mediterranean plant species, including P. lentiscus , J. phoenicea , Myrtus communis L., R. officinalis , Olea europaea L., and Euphorbia dendroides L. They reported that soils associated with P. lentiscus L. exhibited the lowest nitrogen content and the highest abundance of free-living nitrogen-fixing bacteria. In a previous study on the phyllosphere microbiome of P. lentiscus L. collected from the same location utilized for rhizomicrobiome sampling in this study, Abou Jaoudé et al. (2024) highlighted the presence of numerous strains exhibiting a high tolerance to osmotic stress. These findings support the hypothesis that these microbiomes can thrive under similar environmental conditions and may be utilized in microbiome transplantation experiments. The application of the three rhizomicrobiomes showed dissimilarities in sage biomass production and allocation, leaf number and morphology, leaf ecophysiological responses, and leaf metabolome compared to non-inoculated plants. While inoculation did not significantly alter total plant biomass regardless of the treatment, sage inoculated with the microbiome extracted from J. phoenicea exhibited a notable increase in root biomass compared to non-inoculated controls ( Figure 4 ). As Chieb and Gachomo (2023) reported, an increase in root surface in drought-stressed plants can enhance water and nutrient uptake and boost hydraulic conductivity, improving adaptation to water deficit conditions. The observed higher root biomass in sages inoculated with the microbiome extracted from J. phoenicea might be attributed to changes in hormonal signaling. PGPR can interfere with phytohormone signals and control root development ( Ranjan et al., 2024 ). Mainly, auxin is involved in the emission of lateral root and root hairs, promoting nutrient uptake by increasing the root surface ( Rivas et al., 2022 ). Many root-associated microbial strains have been shown to produce auxin ( Keswani et al., 2020 ). Several researchers have demonstrated that the synthesis of this compound is essential for the plant–PGPR interaction, influencing both the phenotypic and transcriptional responses of the host plant ( Spaepen et al., 2007 ; Luziatelli et al., 2020 ; Xu et al., 2023 ). Inoculation with the rhizomicrobiome from J. phoenicea resulted in a reduction in the average leaf area, accompanied by an increase in leaf number to maintain a similar total leaf surface area (see Table 1 ). Individual cells’ size variation mostly depends on vacuole expansion through water uptake ( Forouzesh et al., 2012 ). Consequently, reduced leaf size is generally associated with environments with limited water availability ( Basal et al., 2005 ), as drought stress negatively affects leaf expansion ( Gray and Brady, 2016 ). Smaller leaves possess a thinner boundary layer, promoting convective heat dissipation compared to bigger leaves ( Leigh et al., 2017 ) and inducing faster water losses ( Wang et al., 2019 ), positively influencing plant thermoregulation. Furthermore, similar to the behavior observed in compound leaves, the shedding of smaller leaves may help mitigate the effects of localized water stress. This process can prevent widespread hydraulic failure and minimize biomass loss. The reduction in leaf size observed in plants inoculated with the rhizomicrobiome from J. phoenicea compared to non-inoculated plants indicates an enhanced capacity for water availability, probably triggered by the increase in root biomass. As smaller leaves represent an advantage in arid environments where water conservation is crucial, we can hypothesize a better response of plants inoculated with the rhizomicrobiome from J. phoenicea to drought in the long term. Abate et al. (2021) demonstrated the importance of root hydraulics in drought resistance for Salvia species. They suggest that increased biomass allocation to the root system enhances the accumulation of reserves crucial for post-drought recovery. In our study, the observed modifications in leaf structure and root biomass in plants inoculated with the rhizomicrobiome from J. phoenicea could contribute to a more resilient response to water deficit, facilitating superior recovery and survival in plants inoculated with the rhizomicrobiome from J. phoenicea compared to non-inoculated plants under prolonged drought conditions. These results may explain the reduced leaf mass per area (LMA) observed in plants inoculated with the rhizomicrobiome from J. phoenicea ( Table 1 ), a response contrary to that typically observed in plants under water deficit conditions ( de Dato et al., 2013 ). High leaf mass per area represents a potential adaptation to stressful environments such as those characterized by a Mediterranean climate and is associated with increased leaf thickness and density, reducing mesophyll conductance ( Niinemets, 1999 ; Flexas et al., 2008 ). The decrease in leaf thickness induced by the inoculation with the rhizomicrobiome from J. phoenicea may have shortened the mesophyll pathway for CO 2 to carboxylation sites, thereby increasing mesophyll conductance and mitigating stomatal limitations to photosynthesis. Indeed, stomatal closure is a common and rapid plant defense to preserve water ( Gupta et al., 2020 ): when turgor pressure changes in guard cells, stomatal closure is stimulated ( Osakabe et al., 2014 ). The leaf ecophysiological measurements demonstrated that microbiomes induced a different response to drought in inoculated plants. In sage plants not subjected to inoculation, stomatal conductance showed high values in DAT7 and decreased to about 20% (100 mmol H 2 O m −2 s −1 ) in DAT14, maintaining a constant value in the following 2 weeks ( Figure 5 ). Plants inoculated with the microbiome extracted from R. officinalis exhibited a similar trend ( Figure 5a ). These observations are consistent with the findings of Raimondo et al. (2015) , who reported comparable stomatal conductance trends and values in Salvia grown under similar experimental conditions. Similarly, Savi et al. (2016) observed a significant decline in stomatal conductance of S. officinalis growing in natural ecosystems from June to July and August, followed by an increase in September concurrent with elevated soil water potential. Reductions in stomatal conductance were also reported by Abate et al. (2021) in S. officinalis subjected to different water stress levels and subsequent recovery. Caser et al. (2019) showed that stomatal conductance reduction in Salvia dolomitica subjected to severe drought was associated with increased abscisic acid concentration compared to well-watered plants. In response to drought-induced stress, plants synthesize abscisic acid endogenously. This hormone acts as a signaling molecule and triggers the accumulation of ROS in the cytoplasm of guard cells and of Ca 2+ in the cytosol, reducing turgor and inducing stomatal closure ( Osakabe et al., 2014 ; Liu et al., 2022 ). In our study, the abundance of abscisic acid did not follow the same pattern of stomatal conductance, being higher in non-inoculated and in plants inoculated with the microbiome extracted from J. phoenicea compared to those inoculated with the microbiome from R. officinalis (data not shown) at DAT28, suggesting that Salvia spp. can differently respond to reduced leaf water potential induced by water deficit. The observed stomatal conductance response of S. officinalis is characteristic of anisohydric species, which prioritize maximizing stomatal conductance under high water availability and exhibit less stringent stomatal control than isohydric species ( Raimondo et al., 2015 ). This behavior is attributed to a more moderate induction of abscisic acid biosynthesis under drought stress at the root level, resulting in the maintenance, rather than an increase, of abscisic acid concentration relative to leaf tissue water content ( Gallé et al., 2013 ). Unlike the non-inoculated plants and the plants inoculated with the rhizomicrobiome from R. officinalis , which showed a drastic decrease in stomatal opening at DAT14, plants inoculated with the rhizomicrobiome from J. phoenicea and P. lentiscus exhibited a continuous negative trend, culminating in significantly lower minimum stomatal conductance at DAT28 ( Figures 5b , 6c ). Notably, inoculation with the rhizomicrobiome from J. phoenicea mitigated the decline in stomatal conductance observed in non-inoculated and in plants inoculated with the microbiome extracted from R. officinalis at DAT14 ( Figure 5b ), potentially due to increased water availability resulting from greater root biomass. However, this mechanism does not explain the stomatal response observed in plants inoculated with the rhizomicrobiome from P. lentiscus , which instead exhibited a significant increase in electron transport rate at DAT14 and DAT21 compared to the control ( Figure 6 ). This suggests that inoculation with the rhizomicrobiome from P. lentiscus may alleviate water stress through a different mechanism, independent of root biomass enhancement. Liu et al. (2019) reported similar findings, observing that Sambucus williamsii inoculated with Acinetobacter calcoaceticus X128 exhibited less pronounced reductions in stomatal conductance and assimilation rates compared to non-inoculated plants under drought stress. This observation suggests that the interaction between A. calcoaceticus X128 and S. williamsii triggers a drought-mitigating response. Akhtar et al. (2021) reported increased photosynthetic activity and stomatal conductance in drought-stressed Triticum aestivum inoculated with Bacillus sp. and Azospirillum strains, attributing these effects to enhanced activity of antioxidant enzymes, specifically peroxidase and catalase. Despite the higher CO 2 assimilation rate, no increase in biomass was observed, potentially due to the energy demands associated with the production of secondary metabolites. The analysis of the plant metabolic responses induced by inoculation can give important insights into the mechanisms of increased plant resistance or growth promotion under stress conditions. Data presented in Supplementary Figures S2, S3 and Supplementary Tables S1–S3 demonstrate that rhizomicrobiome transplantation significantly altered the leaf metabolome of sage subjected to water limitation. All three rhizomicrobiomes promoted the accumulation of molecules belonging to phenolic compounds, terpenoids and alkaloids, which can be valuable in regulating the plant response to water-limited conditions ( Kumar et al., 2023 ). Phenolic compounds, specifically phenylpropanoids and flavonoids, deriving from the phenylpropanoid pathway ( Deng and Lu, 2017 ), are plant secondary metabolites that contribute to scavenge ROS produced under drought stress and are, therefore, correlated to plant drought tolerance ( Moradi et al., 2017 ). An increase in phenols with increasing drought stress was reported in Salvia sinaloensis subjected to moderate and severe drought ( Caser et al., 2018 ). Higher polyphenol contents were also observed in S. officinalis under mild and severe water deficits ( Bettaieb et al., 2011 ). Under stress conditions, plants often trade between growth and secondary metabolite production. The accumulation of these metabolites typically coincides with reduced biomass, reflecting a shift in carbon allocation. Resources are diverted toward the synthesis of protective compounds, potentially at the expense of growth processes ( de Abreu and Mazzafera, 2005 ). An increase in phenolic compounds is a reported response observed in both PGPR-inoculated plants ( Mashabela et al., 2022 ) and plants infected with pathogens ( Garcia et al., 2018 ). Flavonoids can contribute to various plant defense responses ( Deng and Lu, 2017 ). Increased flavonoid levels were also observed in plants primed with PGPR and infected with pathogens, serving as signatory biomarkers for induced resistance against pathogens ( Tugizimana et al., 2019 ; Carlson et al., 2019 ; Mhlongo et al., 2021 ). Moreover, flavonoids and phenolic acids are recognized as major secondary metabolites exuded by plant roots ( Mandal et al., 2010 ; Cesco et al., 2012 ). Mashabela et al. (2022) proposed that increased levels of these compounds in leaves could prime plants for enhanced defense responses against pathogens and that the exudation of these secondary metabolites by roots serves as a chemotactic strategy to recruit beneficial microbes, thereby influencing rhizosphere microbiome composition and promoting plant-microbe interactions. Moreover, all three rhizomicrobiomes promoted the accumulation of lipids and terpenoids across several classes, which can be valuable in regulating the plant response to water-limited conditions. An accumulation of these secondary metabolites, precisely monoterpenes and sesquiterpenes has been observed in sage under drought stress ( Nowak et al., 2010 ; Caser et al., 2019 ). The synthesis of highly reduced compounds, like isoprenoids, phenols or alkaloids is pushed during water stress, to counterbalance the massive oversupply of NADPH+H + . Thus, the biosynthesis of alkaloids and monoterpenes, through the consumption of NADPH, may contribute to the decrease in the reducing status of the electron transport chain present during stress conditions ( Yahyazadeh et al., 2018 ). Five compounds accumulated in the leaf metabolome of all the inoculated plants, independently of the type of rhizomicrobiome that was used. These metabolites belonged to five distinct classes, confirming that: (1) the rhizomicrobiome transplanting affects the leaf metabolome at multiple levels; (2) there are some metabolites, whose abundance is specifically altered, that can be used as biomarkers to monitor if the plant has recruited beneficial microorganisms. We found an increase in genkwanin abundance in all inoculated plants. Genkwanin has antibacterial ( Cottiglia et al., 2001 ) and radical scavenging activity ( Kraft et al., 2003 ). An increase in genkwanin was observed in R. officinalis plants grown in the dune sand during the summer, suggesting that the specific synthesis of flavonoids is enhanced in response to environmental stress ( Boscaiu et al., 2019 ). Another up-regulated phenolic compound in inoculated plants is the phenylpropanoid cinnamoyl-beta-D-glucoside, a molecule that derives from a trans-cinnamic acid reacting with a beta-D-glucose ( Deshaies et al., 2022 ). Deshaies et al. (2022) investigated chitosan’s impact on wheat’s early metabolomic response to Fusarium graminearum infection. Their analysis revealed a downregulation of cinnamoyl beta-D-glucoside during infection. As cinnamic acids are precursors to lignans, compounds known to reinforce plant cell walls and hinder fungal penetration, the authors suggest that this downregulation may impair lignification as a defense mechanism against F. graminearum . This result suggests that the increase in the presence of cinnamoyl-beta D-glucoside in our study indicates a potential priming effect of the microbiomes on S. officinalis lignification, which can serve as defense mechanisms against pathogens but can also enhance structural resilience under drought stress ( Choi et al., 2023 ). Among the molecules up-regulated in all inoculated plants, the apocarotenoid beta-ionone has been reported to increase in abundance in plants subjected to salt stress ( Mehdikhanlou et al., 2021 ). Apocarotenoids, products of carotenoid breakdown, are compounds that serve as hormones, volatile aromas, and intracellular secondary messengers ( McQuinn and Waters, 2024 ). These molecules have been reported to be regulators and precursors of protective compounds in response to variations of environmental water, associated with drought tolerance ( Vieira et al., 2024 ). Beta-ionone has been proposed as one of the signals, together with salicylic acid and jasmonate, initiating systemic acquired resistance ( Huded et al., 2023 ). Beta-ionone application in Arabidopsis triggered extensive transcriptomic reprogramming, affecting numerous genes involved in stress responses, growth regulation, hormone metabolism, pathogen defense, and photosynthesis, enhancing resistance to Botrytis cinerea ( Felemban et al., 2024 ). Interestingly, the authors reported that beta-ionone shares many features with another signaling molecule, beta-cyclocitric acid, which elicits plant drought tolerance ( D’Alessandro et al., 2019 ). These results indicate that the upregulation of common metabolites induced by inoculation of the microbiomes can enhance S. officinalis resistance to biotic stress and drought tolerance. In addition to common alteration of the above-mentioned classes and metabolites, inoculation with distinct rhizomicrobiomes also resulted in variations in microbiome-specific classes of compounds and abundance of unique metabolites, underscoring the specialized nature of plant-microbes interactions. Lei et al. (2019) demonstrated that besides the same selecting forces being responsible for the assembly of the core rhizosphere microbiome, the bacterial community composition associated with six plant species is specific to the plant hosts, and the more phylogenetically distant the plant hosts, the more distinct their associated bacterial communities are. These findings can have implications for microbiome selection to enhance the production of exclusive plant metabolites under water shortage, because the targeted application of drought has been proposed as a strategy to improve the quality of medicinal plants ( Selmar and Kleinwächter, 2013 ). Manipulating the plant microbiome may offer a complementary approach to further enhance this effect. More importantly, these metabolites can be used as biomarkers for assessing the establishment of plant-microbiome interactions. The PCA analysis of the leaf metabolome datasets provided further evidence that inoculation with the three microbiomes significantly altered the profiles of detectable leaf metabolites compared to non-inoculated plants ( Figure 2 ). The analysis of the abundance of the top 10 important features that explain PCA differences ( Figure 3 ) showed differences among the treatments. Among the most abundant metabolites, DL-arginine and chromomoric acid concentrations were higher in plants inoculated with the rhizomicrobiome from J. phoenicea compared to all the other treatments. Arginine is accumulated in drought-tolerant clones of eucalyptus trees and sesame genotypes subjected to drought stress ( You et al., 2019 ; Noleto-Dias et al., 2023 ). Moreover, this amino acid was found to reduce the lipid peroxidation in tomatoes under water stress, increasing ascorbate and reducing glutathione, differently from non-treated plants ( Nasibi et al., 2011 ). The foliar application of arginine has been proven to increase endogenous phytohormones (auxins, gibberellins and cytokinins) in wheat while reducing abscisic acid ( El-Bassiouny et al., 2008 ). Vílchez et al. (2018) observed the inoculation of pepper plants under drought stress with Microbacterium sp. 3J1 resulted in changes to the leaf metabolite profile, specifically affecting the molecules’ concentration in regulating osmotic pressure. Notably, the altered metabolites detected in the inoculated plants exhibited a mirrored response to those detected in Microbacterium sp. 3J1 when subjected to drought conditions. Among the metabolites whose abundance increased in inoculated plants, the authors reported arginine; however, it was not upregulated in the microorganism alone when cultivated under water stress. Polyunsaturated fatty acids (PUFA) are essential components of biological membranes, contributing significantly to their structural integrity and fluidity. Moreover, oxygenated PUFA derivatives (oxylipins) serve as bioactive metabolites, that modulate various signal transduction pathways, influencing diverse cellular processes ( Savchenko and Dehesh, 2014 ). Among oxylipins, jasmonic acid (JA) and its immediate precursor, 12-oxophytodienoic acid (OPDA), are the most extensively characterized ( Eckardt, 2008 ). Chromomoric acid is a 12-oxophytodienoic acid metabolite. By the observations documented by Leporino et al. (2024) , which reported an increased level of chromomoric acid B in tomatoes treated with protein hydrolysates, thereby enhancing recovery from drought stress, the modulation of fatty acids in plants inoculated with the rhizomicrobiome from J. phoenicea may have led to a change in membrane composition, consequently influencing cellular redox status. Steviol and amaranthine were reduced in plants inoculated with the rhizomicrobiome from J. phoenicea compared to all the other treatments. In an analysis of the effect of microbial biostimulants on maize metabolism under drought, Othibeng et al. (2022) found steviol glycosides to accumulate in the plant sap. The authors suggest that the microbial biostimulants trigger the active transport of these molecules to other plant tissues, where they are likely hydrolyzed into sugars and steviol, the latter of which can then be converted into gibberellins. Amaranthine is a pigment found in Amaranthus and is known for its bioactive activity. In a study conducted to select Amaranthus genotypes for increased amaranthine content, Gins et al. (2002) found in the enriched cultivar Valentina that amaranthine biosynthesis was negatively correlated to leaf lignin, protein, and cellulose content and leaf density. The authors suggest a link between amaranthine biosynthesis and nitrogen metabolism, potentially with amaranthine as an intermediate in cellular nitrogen compound conversion. These results align with our research, in which a decreased abundance of amaranthine was observed in plants inoculated with the rhizomicrobiome from J. phoenicea , resulting in an increased number of leaves compared to the other treatments. Moreover, as shown in Supplementary Figure S3 , inoculation with the rhizomicrobiome from J. phoenicea significantly increased diterpenoids belonging to the gibberellin class, which are plant hormones that regulate various developmental processes. Both rhizomicrobiomes from J. phoenicea and P. lentiscus stimulated the production of tryptophan alkaloids, classified as simple indole alkaloids, which include auxin-related compounds. Besides, the abundance of compounds belonging to the classes of sphingolipids and steroids, such as sphingosine-1-phosphate, sphinganine-1-phosphate, and 19-hydroxytestosterone ( Supplementary Table S3 ) decreased following inoculation with rhizomicrobiomes from J. phoenicea and R. officinalis . Inoculation with rhizomicrobiomes from J. phoenicea and P. lentiscus resulted in a significant reduction in compounds associated with diterpenoids (3 beta,15,16-trihydroxydolabrene), sesquiterpenoids (artemisinin), and tryptophan alkaloids (pumiloside). A significant reduction in the relative abundance of 19-hydroxytestosterone (−1.25-fold) was also observed in plants inoculated with the rhizomicrobiome from P. lentiscus , and the relative abundance of 3 beta,15,16-trihydroxydolabrene decreased by 1.5-fold in the leaf metabolome of plants inoculated with the rhizomicrobiome from R. officinalis . Compounds related to the four classes mentioned above are involved in defense mechanisms and signaling processes mediating stress responses ( Tholl, 2015 ; Wang et al., 2018 ; Mamode Cassim et al., 2020 ; Liu et al., 2021 ; Mohammadi-Cheraghabadi and Hazrati, 2023 ). Their decrease is part of a more complex alteration in the leaf sage metabolism induced by microbiome transplantation. It can be postulated that the reduction of specific metabolites is associated with increased utilization as precursors for other metabolites or decreased synthesis due to re-routing their precursors toward alternative pathways. Interestingly, the unique inoculum responsible for reducing the relative abundance of all six shared metabolites was the rhizomicrobiome from J. phoenicea . This specific rhizomicrobiome was the only one contributing to increased root biomass ( Figure 4 ). The inoculation with this rhizomicrobiome influenced several metabolic pathways, primarily affecting one compound from each class. The total number of upregulated metabolites was significantly lower than that observed in the leaf metabolome of plants inoculated with the rhizomicrobiomes from P. lentiscus and R. officinalis . Moreover, the results reported in Supplementary Table S2 demonstrated that the inoculation did not result in excessive upregulation, with increases ranging from 2- to 3-fold compared to non-inoculated plants. Inoculation with the rhizomicrobiome from P. lentiscus led to the accumulation of four phenylpropanoids and two flavonoids, with the flavone diosmetin accumulating up to 5.7-fold more than in non-inoculated plants ( Supplementary Table S2 ). In contrast, inoculation with the rhizomicrobiome from RO resulted in the accumulation of seven distinct flavonoids, three of which exhibited increases between 3.7 and 5.1-fold. A significant correlation between the genetic distance of rhizosphere microbial communities and the phylogenetic distance of host plant genotypes was observed ( Bouffaud et al., 2014 ). This indicates that the evolutionary history of a plant genotype influences the selection of bacterial taxa and shapes the rhizosphere microbiota ( Lei et al., 2019 ). We can speculate that R. officinalis -derived rhizometabolome might have triggered a less pronounced response in the closely related S. officinalis compared to J. phoenicea and P. lentiscus microbiomes, highlighting that donor and recipient plant’s phylogeny can influence the response to microbiome transplantation. In evaluating the effectiveness of microbial inoculants in enhancing stress tolerance, certain studies have employed shoot biomass as the sole indicator ( Schmitz et al., 2022 ). However, Monohon et al. (2021) demonstrated that inoculated plants can exhibit reduced biomass despite developing drought-resistant traits. This phenomenon was attributed to a microbially induced drought avoidance strategy, highlighting the potential for morphological changes prioritizing water conservation more than growth. While acknowledging the value of biomass estimation as a metric for evaluating growth promotion in fast-growing species, it is essential to recognize its limitations in accurately reflecting the benefits of microbial inoculation in slow-growing or stress-tolerant species. Moreover, reliance on biomass measurements can be misleading, particularly in short-term experiments. Based on the observations made by Garcia et al. (2018) on potato plants infected with Phytophthora , metabolomics could facilitate the early detection of stress symptoms in asymptomatic plants. The results obtained from this study demonstrate that a comprehensive understanding of plant responses to transplanted microbiomes requires an integrated approach, which includes biomass assessment, physiological analysis, and metabolomics. In cases where biomass remains unchanged, the metabolic adjustments induced by microbial consortia in plants can only be effectively analyzed through a multifaceted approach."
} | 11,479 |
36036292 | PMC9424452 | pmc | 4,148 | {
"abstract": "Natural soil has the ability to suppress the soil-borne pathogen to a certain extent, and the assemblage of soil microbiome plays a crucial role in maintaining such ability. Long-term monoculture accelerates the forms of soil microbiome and leads to either disease conducive or suppressive soils. Here, we explored the impact of soil conditions on bacterial wilt disease (healthy or diseased) under long-term tobacco monoculture on the assemblage of bacterial and fungal communities in bulk and rhizosphere soils during the growth periods. With Illumina sequencing, we compared the bacterial and fungal composition of soil samples from tobacco bacterial wilt diseased fields and healthy fields in three growth periods. We found that Proteobacteria and Ascomycota were the most abundant phylum for bacteria and fungi, respectively. Factors of soil conditions and tobacco growth periods can significantly influence the microbial composition in bulk soil samples, while the factor of soil conditions mainly determined the microbial composition in rhizosphere soil samples. Next, rhizosphere samples were further analyzed with LEfSe to determine the discriminative taxa affected by the factor of soil conditions. For bacteria, the genus Ralstonia was found in the diseased soils, whereas the genus Flavobacterium was the only shared taxon in healthy soils; for fungi, the genus Chaetomium was the most significant taxon in healthy soils. Besides, network analysis confirmed that the topologies of networks of healthy soils were higher than that of diseased soils. Together, our results suggest that microbial assemblage in the rhizosphere will be largely affected by soil conditions especially after long-term monoculture. Supplementary Information The online version contains supplementary material available at 10.1186/s13568-022-01455-1.",
"introduction": "Introduction Bacterial wilt, caused by Ralstonia solanacearum , is one typical soil-borne disease and can bring severe losses to agricultural crops (Genin and Denny 2012 ; Jiang et al. 2017 ). In addition, long-term monoculture is more likely to cause rapid accumulation of R. solanacearum in soil (Chen et al. 2020a ). Until now, there is currently no effective chemical pesticide to manage this disease (Liu et al. 2013 ). As an attractive alternative, antagonistic microbes were introduced as potential biocontrol agents (Guo et al. 2014 ). A number of antagonism studies have dealt with the pairwise interactions between beneficial and pathogenic microbes (reviewed by de Boer 2017 ). Unfortunately, in real communities, the complexity of the soil environment may largely decrease their inefficiency of pathogen suppression (Mallon et al. 2015 ; de Boer 2017 ). However, the phenomenon of ‘disease-suppressive soils’ shows an ideal model by which plant protection can be triggered by soil microbes (reviewed by Wang and Li 2019a ). Hence, a better knowledge of understanding the positive functions from indigenous microbial communities in the soil is essential for a sustainable and effective bacterial wilt management strategy. Indigenous microbial communities in the soil forms complex networks and manipulates plant health (Berendsen et al. 2012 ). Based on the composition of the resident microbial community, a biological barrier is formed, with microbes interacting with pathogens and defending against invasion by pathogens near the root surface (Raaijmakers et al. 2009 ; Fu et al. 2017 ). Therefore, assessing the relationship between the soil community and crop morbidity is a critical step toward understanding potential impacts of these communities on plant health (Rosenzweig et al. 2012 ; Cha et al. 2016 ; Xiao et al. 2018 ). It has been revealed that species-rich biomes are more resistant than species-poor biomes to pathogen invasions (Wei et al. 2015 ) and high incidence of soil-borne diseases could be due to the deterioration of the soil microecological environment (Gao et al. 2020 ). Additionally, tobacco farmlands with high biodiversity were more resistant to pathogen infection (Wang et al. 2019 ). Soil microbial community changes dramatically during plant growth (Lundberg et al. 2012 ; Xiong et al. 2015 ). It is important to understand the composition and interaction of microbial communities during plant development (Chaparro et al. 2014 ). Evidence suggests that Arabidopsis at different developmental stages can culture specific rhizosphere microbiome members (Yuan et al. 2015 ). Similarly, the rhizosphere microbiome characteristics of maize change with growth stage (Li et al. 2014 ). During infection by bacterial wilt, the composition of the microbial communities in the rhizosphere of tomato at different growth stages is significantly different (Wei et al. 2018 ). Research has demonstrated that plant is a unique determinate of community structure in the rhizosphere at early stages, but that these differences in the microbiome disappear as plant develops (Inceoğlu et al. 2011 ). Here we report the results of the bacterial wilt diseased and healthy soil microbial assemblages at different growth stages of tobacco. We included the soils collected from diseased fields, and the ones from healthy fields. Bulk soils were collected in March and rhizosphere soils were collected in July and September. We examined the soil biochemical properties and microbial compositions. We investigated the influences of soil conditions and tobacco growth periods on the bacterial and fungal assemblage.",
"discussion": "Discussion Here, we investigated bacterial and fungal communities from the early growth stage to the last two growth periods and the microbial community in the later stages of tobacco growth plays an integral role in plant-pathogen interactions. Increasing evidence has shown that the rhizosphere microbial community plays an indispensable role in relieving nutrient stress and responding to pathogenic micro-invasion by using root exudates from plant roots (Okubo et al. 2016 ). Plants are able to recruit specific bacteria and fungi for defense against bacterial wilt in the rhizosphere (Lareen et al. 2016 ). Additionally, the specific selection of microbiome by plants in the rhizosphere mainly differs at different developmental stages (Yang and Crowley 2000 ; Bulgarelli et al. 2012 ). Infection by pathogenic bacteria is the main cause of plant recruitment of beneficial microorganisms in the rhizosphere (Bakker et al. 2013 ), and the antagonistic effect on pathogens is enhanced during plant development (Hu et al. 2020 ). Specific resident plant rhizosphere bacterial communities that adapt to plants play important roles in both optimize growth and protecting against infection by pathogens. The recruitment of beneficial microorganisms can also change the physiological function of plants to allow them to resist aerial pathogens (Kumar et al. 2012 ). Although the rhizosphere effects on microbial assemblage is proved to be crucial in plant health, it also have been reported that the initial variation in soil bacterial composition and functioning can determine the outbreak of bacterial wilt disease (Wei et al. 2019 ). And thus, understanding the difference of microbial community in healthy and diseased soils are important regarding to plant-pathogen interactions. In this study, we confirmed significant shifts in the diversity and abundance of bacterial and fungal communities associated with healthy and diseased soils. Indeed, there are increasing studies focusing on the microbial indicators associated with the suppression of tobacco bacterial wilt (Liu et al. 2016 ; She et al. 2017 ). Here, we used LEfSe and co-occurrence network to investigate the keystone species as well. Network analysis have been widely used to determine the association and co-occurrence complexity of microorganisms (Su et al. 2020 ). Our study shows that the rhizosphere soil co-occurance network of healthy tobacco plants is more complicated than that of diseased tobacco plants. This is consistent with previous research results (Yang et al. 2017 ). Indeed, microbial communities with relatively high diversity have better resistance to invasion by pathogenic bacteria (Hu et al. 2020 ). Interactions between microbial species can affect disease dynamics by changing the relative and absolute density of pathogens in the host-associated microbiome (Mendes et al. 2011 ; Mueller and Sachs 2015 ). In the healthy and diseased rhizosphere soil networks, we observed positive interactions between nodes, indicating niche overlap, as well as negative interactions, which suggest competition or exclusion (Faust and Raes 2012 ). Competitive interactions and the production of antimicrobial compounds play an important role in controlling pathogen density and disease dynamics (Wei et al. 2015 ). Further experimentation is needed to decipher the impact of competitive microbes on soil microbial ecological networks and plant health. The greater variety of potential key taxa observed in the rhizosphere samples might be beneficial to maintain plant health. Analyses of LEfSe and network analysis showed that Flavobacterium and Pseudomonas may be the most active microbial species in healthy soil. Flavobacterium can play a role in biological control by producing antibacterial effect factors, antibacterial substances, extracellular macromolecular degrading enzymes, etc. (Bernardet and Nakagawa 2006 ; Kwak et al. 2018 ; Carrion et al . \n 2019 ). Pseudomonas which can produce antifungal/inhibitory compounds and siderophores that can control against bacterial wilt disease (Ramesh and Phadke 2012 ; Chandrasekaran et al. 2016 ). High Pseudomonas diversity can reduce R. solanacearum density in the rhizosphere and decrease the disease incidence due to both intensified resource competition and interference with the pathogen (Hu et al. 2016 ). Notably, the network analysis revealed the Mortierella and Fusarium were key species in healthy soils. It was also reported from previous studies that Mortierella was an indicator species in disease suppressive soils (Expósito et al. 2017 ; Xiong et al. 2017 ). Mortierella can produce antibiotics, and has potential antagonist activity against various plant pathogens (Tagawa et al. 2010 ). F. oxysporum confers biocontrol against root diseases in various plants (Lamo and Takken 2020 ). Thus, a potentially beneficial microbiome may form cooperative associations with other taxa to maintain plant health. In conclusion, our results showed that there are significant differences in microbial composition between healthy and diseased soils. Both factors of soil conditions and tobacco growth periods can have an influence on the bulk and rhizosphere microbial composition. Yet, the impact of soil conditions is larger than that of tobacco growth periods in the rhizosphere soils. Discriminative taxa determined by LEfSe and network analysis in healthy soils showed beneficial potentials. This implies that steering soil microbiome in a beneficial way could have great opportunities to maintaining soil-borne disease. However, these findings need to be further confirmed in greenhouse experiments."
} | 2,799 |
38638154 | PMC11025542 | pmc | 4,150 | {
"abstract": "The microbial communities, inhabiting around and in plant roots, are largely influenced by the compartment effect, and in turn, promote the growth and stress resistance of the plant. However, how soil microbes are selected to the rhizosphere, and further into the roots is still not well understood. Here, we profiled the fungal, bacterial communities and their interactions in the bulk soils, rhizosphere soils and roots of eleven stress-resistant plant species after six months of growth. The results showed that the root selection (from the rhizosphere soils to the roots) was stronger than the rhizosphere selection (from the bulk soils to the rhizosphere soils) in: (1) filtering stricter on the fungal (28.5% to 40.1%) and bacterial (48.9% to 68.1%) amplicon sequence variants (ASVs), (2) depleting more shared fungal (290 to 56) and bacterial (691 to 2) ASVs measured by relative abundance, and (3) increasing the significant fungi-bacteria crosskingdom correlations (142 to 110). In addition, the root selection, but not the rhizosphere selection, significantly increased the fungi to bacteria ratios (f:b) of the observed species and shannon diversity index, indicating unbalanced effects to the fungal and bacteria communities exerted by the root selection. Based on the results of network analysis, the unbalanced root selection effects were associated with increased numbers of negative interaction (140 to 99) and crosskingdom interaction (123 to 92), suggesting the root selection intensifies the negative fungi-bacteria interactions in the roots. Our findings provide insights into the complexity of crosskingdom interactions and improve the understanding of microbiome assembly in the rhizosphere and roots.",
"conclusion": "Conclusion Our study describes the effects of the the rhizosphere and root selections on structuring the fungal and bacterial communities in bulk soils, rhizosphere soils, and roots of a group of stress-resistant plant species. Both of the two selections intensify the crosskingdom fungi-bacteria interaction. Compared to the rhizosphere selection, the root selection is more intensive and unbalanced by accumulating more crosskingdom and negative interactions.",
"introduction": "Introduction The plant underground area, including plant roots and their surrounding soils, is abundantly colonized by different microbes, especially by abundant fungal and bacteria species ( Mitter, De Freitas & Germida, 2017 ; Zhalnina et al., 2018 ). According to the physical proximity to the plant surface and the level of host influence on microbial communities, this area is separated to two different compartments: the rhizosphere and the root endosphere ( Fitzpatrick et al., 2018 ; Yurgel et al., 2018 ). It is supposed that the plant deploys two layers of selection to recruit and assemble the microbial communities inhabiting in these two compartments: the rhizosphere communities are formed with microbes mainly from the bulk soils, while the root endosphere communities are formed with microbes mainly from the rhizosphere ( Reinhold-Hurek et al., 2015 ). Both of the two layers of selection are affected by plant genetics, and in turn, the assembled microbial communities can deliver essential ecosystem services, such as nutrient cycling, soil structuring, and stress alleviation, back to the plant ( Saleem et al., 2018 ). The plant rhizosphere selection is mainly achieved by root exudates, such as many small signaling molecules ( Hassan & Mathesius, 2012 ; Huang et al., 2014 ), polymers ( Beauregard et al., 2013 ), antimicrobials ( Huang et al., 2014 ), and plant hormones ( Lebeis et al., 2015 ). In comparison, the root selection is mainly achieved by plant epidermis and plant immunity system ( Reinhold-Hurek et al., 2015 ). Both of the selections can alter plant-microbe and microbe-microbe interactions ( Dundore-Arias et al., 2023 ; Zhang et al., 2024 ), thus cause the microbial taxal and functional variations to improve plant health and stress resistance ( Yeoh et al., 2017 ; Cordero, de Freitas & Germida, 2020 ). Different microbial interaction types can have different biological implications to their host plant ( Chepsergon & Moleleki, 2023 ; Liu et al., 2023a ). For example, the positive interactions, generally recognized as microbial cooperation, of different fungal species were capable to improve metabolic efficiency in the root endosphere and rhizosphere soils of the plants Miscanthus sinensis and M. floridulus ( Ji et al., 2023 ). The negative interactions, generally recognized as microbial competition, of bacteria in competition for iron are efficient in suppression of potential soil pathogens in the rhizosphere of various crops ( Gu et al., 2020 ). In response to environmental stresses and biotic pathogens, the frequency of microbial interactions generally increases, and confers beneficial effects to improve plant resistance ( Ge et al., 2023 ; Zhou et al., 2023 ). Many of these microbial interactions are predictable, and can be characterized and synthesized to form brief communities for ecological and agricultural applications ( Wu et al., 2023 ). However, the microbial assembly and interactions of most plants, especially those plants with specific ecological and agricultural attributes, are still not completely studied. In this study, eleven plant species were selected. All these plant species are native plants in Hainan, China, which can adapt to the harsh environment of soil high salinity and alkalinity, high temperature, strong light, and frequent droughts ( Tong et al., 2013 ; Liu et al., 2015 ; Ren et al., 2017 ; Li et al., 2018 ). Furthermore, several of these plant species have been experimentally proved with strong abilities in improving soil water and nutritional conditions, and also in resistance to abiotic stresses ( Wang et al., 2019 ; Zhang et al., 2019 ; Li et al., 2021 ). Based on these abilities, these plant species have been recommended and used as pioneer plants in restoration of destroyed and degraded ecosystems ( Tong et al., 2013 ; Liu et al., 2015 ; Ren et al., 2017 ). Our study focused on the underground fungal and bacterial communities associated with these plant species, and aimed to test whether the soil fungal, bacteria communities, and their crosskingdom interactions respond differently to the rhizosphere and root selections.",
"discussion": "Discussion Hainan Island, with 33, 900 km 2 of land area, is the second largest island in China. It locates in the zone of tropical monsoon climate, with an annual average temperature of 23.2–27.1 °C, annual total precipitation of 1,009–2,367.7 mm (nearly 70% in the wet season), and annual sunshine duration of 1,776–2,783 h ( Chen et al., 2022 ). For the selected plant species in this study, their native growing soils mainly consisted of phospho-calcic soils and coastal saline soils, which are usually saline, alkaline, and poor in content of organic carbons ( Zhou, Zhou & Wang, 2003 ; Li et al., 2018 ). Based on their great adaptation to barren lands, these plant species have attracted more and more attentions in the past several years in China ( Li et al., 2018 ). For example, Wang et al. (2019) demonstrated that the growth of one or several of these plants improved soil quality and vegetation recovery rate by significant increasing of soil contents of water, microbial biomass carbon and nitrogen. Most of all, the plants of Calophyllum inophyllum and Guettarda speciosa showed strong abilities of stress resistance ( Wang et al., 2019 ; Zhang et al., 2019 ; Li et al., 2021 ). However, their root microbiomes, supposed to offer many beneficial traits to plant growth and resistance, have received little attention. Our study offered a snapshot of the assembly of fungal and bacterial communities after the abiotic stress-resistant plants growth for about six months. It proved that both fungal and bacterial communities respond more drastically to the root selection than the rhizosphere selection ( Reinhold-Hurek et al., 2015 ). The shared fungal and bacterial ASVs were more frequently depleted by the root selection than by the rhizosphere selection ( Fig. 2 ). With abundant root exudates in the rhizosphere area, many different microbes can co-exist and work synergistically to transform unusable substrates to usable substrates and reduce toxic substances ( McLaughlin et al., 2023 ; Wu et al., 2023 ). The usable substrates can further support the growth of many microbial saprophytes, which results in the relatively more abundant fungal and bacterial communities in the rhizosphere ( Hassan & Mathesius, 2012 ; Trivedi et al., 2020 ). In contrast, the root endosphere is protected by a physical barrier (root epidermis) and the plant defense system, which results in the relatively less abundant fungal and bacterial communities in the root ( Hassan & Mathesius, 2012 ; Trivedi et al., 2020 ). In addition, the priority effect, namely, early colonized microbes affect the colonization of microbes that arrive later, may also affect the abundances of fungal and bacterial communities in the root ( Debray et al., 2022 ). Furthermore, we found that the fungi to bacteria ratios (f:b) of both the observed species and shannon diversity index were significantly increased by root selection other than by rhizosphere selection ( Figs. 1C and 1D ). These results suggest that: (1) root selection may have weaker effects on fungal communities and/or stronger effects on bacterial communities; (2) root selection may facilitate the growth of fungal endophytes which can inhibit the growth of some bacteria. Many fungal endophytes can establish a mutualistic relationship with plant roots, in which the fungi can improve the growth and stress resistance of their host plant through mechanisms such as phosphorus transmission, stimulation of proline, glycine betaine production, increase of plant indoleacetic acid concentration etc . ( Liu et al., 2023b ; Miranda et al., 2023 ; Toppo et al., 2023 ). In a recent study, Ma et al. (2023) showed that the mutualistic growth of a fungal endophyte ( Phomopsis liquidambaris ) in the plant ( Arachis hypogaea L.) roots not only promoted host growth and disease control, but also reshaped the core root bacterial taxa. Based on these, one explanation of the unbalanced effects of root selection on fungal and bacterial communities may be that the roots of our stress-resistant plants associate closely with their endosphere fungal communities, and then, some fungal endophytes reshape the bacterial communities through crosskingdom interactions. Based on the correlation and network analysis, we found that the microbial total interaction increased in number and strength in response to the rhizosphere and root selections ( Figs. 3 and 4 ). Firstly, we found that all the microbial communities of bulk soils, rhizosphere soils, and roots were dominated by positive interactions. This may be explained by that the microbial communities rely on cooperative relationships to improve metabolic efficiency and promote soil and root colonization ( Chepsergon & Moleleki, 2023 ; Ge et al., 2023 ; Zhang et al., 2024 ), such as the microbial interaction forms of crossfeeding, syntrophy, physical complex formation, etc . ( Wu et al., 2023 ). In addition, some of the positive interactions may also be caused by spatial coincidence, rather than actual ecological interactions ( Liu et al., 2023a ). Secondly, we found that the increase of negative interactions was consistent across the fungal, bacterial, and the combined microbial communities in response to both of the two selections. Negative interactions, may be representative of microbial competition, antagonism, or predation, are important for the ecological stability of a microbial community ( Ji et al., 2023 ; Liu et al., 2023a ). In bulk and rhizosphere soils, the maintenance of community positivity (more positive interactions) is beneficial for the microbial community to transform transient root exudates and scavenge soil pathogens and hazardous substances ( Ji et al., 2023 ; Li et al., 2023 ; Zhou et al., 2023 ). While in plant roots, the longer ecological stability, by increased negative interactions, may be important for the establishment of mutualistic plant-microbe relationships ( Dundore-Arias et al., 2023 ; Ji et al., 2023 ). Lastly, we found that the increased number of negative interactions of the combined microbial community correlated to the increased number of inter-kingdom interactions by the root selection ( Figs. 5D and 5E ). Negative interactions have been proved to primarily occur through inter-kingdom microbial interactions, and are important for plant host survival and maintenance of host-microbiota balance ( Chen et al., 2018 ; Durán et al., 2018 ; Zhang et al., 2024 ). The competition between fungal and bacterial species in plant roots may be caused by their common antagonistic relationships and metabolic overlaps ( Pacheco & Vorholt, 2023 ; Zhang et al., 2024 ). Based on these, we infer that the increase of both the numbers of negative interactions and inter-kingdom interactions causes the unbalanced influence of the root selection on fungal and bacterial communities. However, more experimental works are still needed to conform these inferences, and to recur the fungi-bacteria interactions for agricultural and ecological applications."
} | 3,345 |
38410456 | PMC10896350 | pmc | 4,154 | {
"abstract": "Horizontal gene transfer (HGT) is a fundamental process in the evolution of prokaryotes, making major contributions to diversification and adaptation. Typically, HGT is facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages that generally impose fitness costs on their hosts. However, a substantial fraction of bacterial genes is involved in defense mechanisms that limit the propagation of MGEs, raising the possibility that they can actively restrict HGT. Here we examine whether defense systems curb HGT by exploring the connections between HGT rate and the presence of 73 defense systems in 12 bacterial species. We found that only 6 defense systems, 3 of which are different CRISPR-Cas subtypes, are associated with the reduced gene gain rate on the scale of species evolution. The hosts of such defense systems tend to have a smaller pangenome size and harbor fewer phage-related genes compared to genomes lacking these systems, suggesting that these defense mechanisms inhibit HGT by limiting the integration of prophages. We hypothesize that restriction of HGT by defense systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and fitness effect of HGT in bacterial populations.",
"introduction": "Introduction Bacterial viruses, known as bacteriophages (phages, for short), are the most abundant entities in the biosphere ( Keen, 2015 ). They regularly attack and predate on bacterial populations across different ecological settings with the estimated rate of infection per second in oceans alone is on the order of 10 23 ( Suttle, 2007 ; Mushegian, 2020 ). To counteract phages and other parasitic mobile elements, bacteria evolved a wide range of defense systems with various molecular mechanisms of action ( Doron et al., 2018 ; Gao et al., 2020 ; Bernheim et al., 2021 ; Millman et al., 2022 ; Georjon and Bernheim, 2023 ). These include CRISPR-Cas systems, which provide adaptive immunity by storing information about past encounters with MGE ( Makarova et al., 2020 ), restriction-modification (RM) systems that degrade foreign genetic material based on specific molecular patterns ( Wilson, 1991 ), abortive infection mechanisms that limit the spread of phages in the bacterial population by inducing suicide of infected cells ( Lopatina et al., 2020 ), and multiple others. Individual bacterial genomes typically encode several diverse defense systems, and the repertoire of defense mechanisms can differ even among closely related strains ( Bernheim and Sorek, 2020 ; Tesson et al., 2022 ). Consequently, defense systems demonstrate high mobility, with high rates of gene gain and loss on a short evolutionary scale ( Makarova et al., 2013 ; Puigbo et al., 2017 ). Although defense systems are essential for protection against phages, and to a lesser extent, against other invasive MGEs, such as integrative conjugative elements (ICE) and plasmids, they also come with associated fitness costs to the hosts. One form of such costs is impediment to lysogenic conversion and gain of beneficial genes that reside in MGEs. These include genes that equip bacteria with the capability to adapt to different ecological niches ( Kelleher et al., 2017 ; Davray et al., 2021 ; Kieft et al., 2021 ), and resist environmental stress ( Lopatkin et al., 2017 ; Jahn et al., 2019 ). For example, the presence of CRISPR-Cas system in Enterococcus faecalis shows a significant inverse correlation with the resistance to different antibiotics ( Palmer and Gilmore, 2010 ). Moreover, multiple experimental studies have demonstrated the capability of CRISPR-Cas systems to limit horizontal gene transfer (HGT) ( Marraffini and Sontheimer, 2008 ; Bikard et al., 2012 ). However, broader comparative genomic analyses yielded conflicting conclusions on the inhibition of HGT by CRISPR-Cas on a larger evolutionary scale ( Gophna et al., 2015 ; Shehreen et al., 2019 ; Wheatley and MacLean, 2021 ). Furthermore, potential interference of other defense systems with HGT has not be comprehensively analyzed. In this work, we examined the association between the presence of various defense systems and the rates of gene gain in a set of 12 bacterial species. Our results reveal significant association with increased gene gain rate for 15 defense systems, whereas 6 systems were found to be significantly associated with reduced gene gain rates. However, we show that for the 15 defense systems associated with increased gene gain rates, this signal is likely a byproduct of their location within large MGEs. Conversely, 3 of the 6 defense systems that are negatively correlated with the gene gain are CRISPR-Cas variants that tend to inhibit gene gain by reducing prophage integration.",
"discussion": "Discussion Experiments have clearly demonstrated that bacterial defense systems can actively limit propagation of MGE such as phages and plasmids under laboratory conditions ( Marraffini and Sontheimer, 2008 ; Dupuis et al., 2013 ; Deep et al., 2022 ). However, whether on the larger evolutionary scale these systems provide any substantial barrier to the horizontal gene acquisition, remains a conflicting topic ( Gophna et al., 2015 ; Wheatley and MacLean, 2021 ). Our results indicate that the majority of the defense systems are not associated with either an increased or a decreased rate of gene acquisition. However, a minority of the defense systems are associated with an elevated gene gain rate, and an even a smaller subset is associated with a reduced gene gain rate. While certain CRISPR-Cas systems, Gabija and RM-Type II are associated with a reduced gene gain rate in some species, in other species, they do not exhibit such association. Thus, the effects of defense systems on HGT appear to be strongly lineage-specific and depend on additional factors. Integrated phages and plasmids can impose various metabolic and other fitness costs on their hosts contingent upon ecological and genetic contexts ( Alonso-Del Valle et al., 2021 ; Rendueles et al., 2023 ). Hence, there is likely a differential selection pressure on same types of defense systems in different species to hinder the spread of MGEs, depending on the associated costs of the latter. Perhaps, more generally and more importantly, given that HGT is the major route of acquisition of novel traits by bacteria, the active restriction of the gene flow can compromise bacterial adaptation to diverse, fluctuating environmental conditions ( Woods et al., 2020 ; Arnold et al., 2022 ). In this scenario, the costs associated with defense systems can outweigh their benefits, leading to a reduction in their activity or even complete inactivation and subsequent loss. Furthermore, the extent of HGT varies among different bacterial species and strains depending on the ecological conditions ( Smillie et al., 2011 ; Groussin et al., 2021 ). For example, niche specialists that occupy stable environmental habitats tend to have closed pangenomes and lower genetic diversity relative to generalists with open pangenomes ( Brockhurst et al., 2019 ). As a result, for some bacteria, ecological barriers to HGT could be so pronounced that the restriction of HGT by defense systems becomes limited and could be neither statistically significant nor biologically relevant. Bacteria rely on different molecular strategies, including innate immunity, adaptive immunity and abortive infection to combat infection by phages and restrict the invasion of other costly MGEs, such as conjugative plasmids ( Makarova et al., 2021 ). Such multilayered defense organization provide cells with an enhanced capability to withstand assaults by diverse MGEs. Abortive infection is typically used by bacteria as a last-resort defense strategy during the final stages of phage reproduction when the cell lysis becomes imminent ( Lopatina et al., 2020 ; Rousset and Sorek, 2023 ). Therefore, integration of prophages or uptake of conjugative plasmids are not likely to trigger this type of immune response, and consequently, abortive infection appears unlikely to substantially interfere with HGT facilitated by phages and other MGEs. Indeed, among the 6 defense systems we found to be associated with a reduced gene gain rate, none is known to be involved in abortive infection response. By contrast, 3 of these defense systems are CRISPR-Cas variants from Pseudomonas aeruginosa , Klebsiella pneumoniae and Streptococcus pyogenes that appear to restrict gene gains, primarily, by interfering with prophage integration. On the other hand, we found no evidence that CRISPR-Cas-I-E in Escherichia coli and Salmonella enterica impacts gene acquisition on the species-level, which is consistent with prior work demonstrating the low activity of these systems ( Westra et al., 2010 ; Shariat et al., 2015 ). Our observations of a positive association with some defense systems with HGT rate seem to be explained away by their hijacking of MGE for. Nevertheless, bona fide stimulation of HGT by defense systems cannot be ruled out. For example, in Petrobacterium atrosepticum , CRISPR-Cas-I-F can promote HGT through generalized transduction by boosting the survival rate of cells that receive transduced genetic material during infection, while the defense system inhibits lytic phages ( Watson et al., 2018 ). Such mechanism could represent an adaptation to facilitate the gene flow while maintaining an active defense system that deters deleterious MGEs. Consequently, the interplay of population level dynamics among various MGEs and bacteria that harbor defense systems can determine the varying effects of defense systems on HGT."
} | 2,414 |
36224210 | PMC9556595 | pmc | 4,155 | {
"abstract": "Most cave formation requires mass separation from a host rock in a process that operates outward from permeable pathways to create the cave void. Given the poor solubility of Fe(III) phases, such processes are insufficient to account for the significant iron formation caves (IFCs) seen in Brazilian banded iron formations (BIF) and associated rock. In this study we demonstrate that microbially-mediated reductive Fe(III) dissolution is solubilizing the poorly soluble Fe(III) phases to soluble Fe(II) in the anoxic zone behind cave walls. The resultant Fe(III)-depleted material (termed sub muros ) is unable to maintain the structural integrity of the walls and repeated rounds of wall collapse lead to formation of the cave void in an active, measurable process. This mechanism may move significant quantities of Fe(II) into ground water and may help to explain the mechanism of BIF dissolution and REE enrichment in the generation of canga. The role of Fe(III) reducing microorganism and mass separation behind the walls (outward-in, rather than inward-out) is not only a novel mechanism of speleogenesis, but it also may identify a previously overlooked source of continental Fe that may have contributed to Archaean BIF formation.",
"introduction": "Introduction The tropical regions of Brazil, including Carajás, Iron Quadrangle (IQ), and Southern Espinhaço Range, contain some of the most extensive landscapes of Proterozoic iron deposits in the world 1 , 2 . The upper sequence is comprised of a laminated quartz-hematite banded iron formation, known as itabirite or jaspilite, which hosts some of the largest iron ores deposits in the world 3 , 4 . The formation of these ore bodies, which represent a heterogenous mix of hematite, and goethite, and other Fe-hydroxides, is not clearly understood, but required silica removal followed by Fe replacement/deposition, with a total Fe content up to 67 wt% 2 , 3 , 5 , 6 . These high-grade ores, which are generally low in P, Al and Si, are among the most economically important Fe deposits in the world, representing > 20% of global iron reserves 2 , 7 , 8 . The iron-rich landscapes and subsurface features in Brazil are covered in a ferruginous duricrust known as canga (derived from the indigenous word itapanhoacanga 9 ), which protects the more friable BIF and ore deposits from weathering 1 , 2 , 9 – 13 . This duricrust, which can range in thickness from a few centimeters to 30 m (average 3 m) is composed of detrital fragments of BIF cemented primarily by Fe-oxides, including hematite, goethite, and relatively poorly-crystalline Fe(III) (hydr)oxides 1 , 9 , 10 , 14 , 15 . This cementation generates a well indurated material surface that is extremely resistant to weathering, with rates of 0.17–0.54 m Myr −1 reported 10 , 12 , 16 . Similar to the Fe ores, the mechanism of canga formation, which includes enrichment of P oxides and rare earth elements (REEs) compared to itabirite, is poorly understood 9 , 17 . Canga does not exist as a separate bedded layer, but rather lays over the BIF landscape like a thick blanket, which led Dorr (1964) to suggest that there must be continuous turnover, otherwise it would have been removed thorough denudation long ago, exposing the friable, underlaying BIF. While canga is still much younger than BIF, it has been dated to ~ 65 Mya, making it one of the oldest exposed landscapes in Brazil 9 , 10 , 18 . Nonetheless, the age of canga is not homogenous, and dating has revealed that younger canga is found at depth in the same sampling cores 9 , 10 , 18 . In recent years, there has been increasing evidence that microbial Fe-cycling may be responsible for the maintenance of canga, with Fe(III) reduction releasing Fe(II) that is then oxidized as it moves toward the surface 9 , 12 , 13 , 19 , 20 . The abundance of consolidated Fe(III) (hydr)oxide cements at the surface make it resistant to weathering and limits water infiltration; however, discontinuities in the surface of the canga, such as tension joints, fractures and penetration by plant roots allow water to enter the subsurface, where the crust-like surface give ways to a high-porosity matrix in the canga, with an internal porosity up to 29% 1 , 11 , 21 – 23 . The routes for water into the subsurface and relatively high internal porosity of canga results in the formation of regionally significant aquifers, and water flow can reach 2.80 × 10 –4 m s −1 , comparable to highly fractured rocks and even karst aquifers, with primary porosity occurring at the canga-BIF interface 1 , 11 , 24 . Despite this porosity, the weathering-resistant nature of canga would suggest that karstification is limited; however, these iron landscapes represent some of the most cave-dense regions of Brazil, containing over 3,000 documented iron formation caves (IFCs), representing ~ 20% of all the known caves in Brazil 1 , 21 , 22 , 25 , 26 . Brazilian IFCs were first described in 1818, and remained a relative curiosity until 1988, when the new Brazilian constitution included caves as a natural resource that required a preservation zone 1 , 2 , 25 , 27 . This occurred during a significant increase in mining activities in Brazil, which have grown from ~ 1.5 million tons annually in the 1950s to approximately 20% of world production today (~ 400 million tons annually) 28 . Ore extraction has primarily occurred through opencast mining, and due to the preservation of identified IFCs, has necessitated a meandering pattern across the landscape, impacting ecosystems through a combination of habitat loss and the impact of mining waste effluent 27 – 30 . Most IFCs are short, averaging 30 m in length with an average 2 m diameter 1 . A mechanism of formation of these IFCs was put forward by Simmons 25 , who postulated that IFCs formed due to dissolution by Fe(III) reducing microorganisms (FeRM) or through solubilization of dolomitic cements within the Itabirite. In support of this hypothesis, Parker et al. 31 cultured FeRM from IFC sediments and demonstrated their ability to reduce Fe(III) phases within BIF and canga 31 , 32 . Microbial Fe(III) reduction by cave-asssociated microorganisms was driven by fermentative organisms, which demonstrated pitting of Fe(III) (hydr)oxide surfaces 31 . While fermentation has not previously been associated with large-scale Fe-reduction, Parker et al. 31 , 32 demonstrated that it dramatically accelerated Fe-reduction when compared to respiratory Fe-reduction. Nonetheless, the reduction experiments of Parker et al. 31 , 32 were carried out in batch incubations, where passivation of Fe-oxides by Fe(II) and the closed system could reduce Fe-reduction rates and influence the drivers of FeRM metabolism 31 , 33 . To better reflect the conditions experienced in canga, we demonstrated that under flow conditions bacterial fermentation Fe-reduction led to an accelerated rate of dissolution, which enhanced permeability 33 . Together these data suggested that cave formation processes are driven by water bringing organic carbon from surficial primary productivity into the canga via the surface recharge zone through surface unconformities. Then FeRM activities reduce insoluble Fe(III) (hydr)oxides to relatively soluble Fe(II), which can then be transported via the developing aquifer 1 , 26 , 31 , 33 . Such activity consolidates into flow paths that coalesce around cave conduits, with a mechanism of speleogenesis similar to the mass separation and transport seen in other karst systems, albeit driven by FeRM 1 , 31 , 34 ; however, there are limitations in this model, as Fe-reduction cannot occur in the presence of oxygen (making cave formation difficult to reconcile with a conduit model) and a conduit model does not match the morphology of the observed IFCs (intercalating rooms, carved floors, no relationship with lithology) 1 . In this work, we reconcile these observations with FeRM activity using a combination of techniques in geology, materials chemistry, and geomicrobiology, to demonstrate that Fe-reduction is occurring behind the walls of the cave, sequestered from atmospheric O 2 . We demonstrate that this FeRM activity leads to extensive Fe(III)-reduction in situ, promoting passage collapse and enlargement, in an active and ongoing process that matches the observed morphology 1 . The low pH and anoxic conditions, along with the presence of apparent electron shuttles, may explain the enrichment of P oxides and REEs in canga 9 . These data demonstrate not only a novel method of cave formation, but suggest a more significant mechanism of subsurface Fe mobilization and weathering than has previously been considered 35 – 37 .",
"discussion": "Discussion Our goal has been to understand the processes that lead to the formation of IFCs in the relatively impermeable iron landscapes of Brazil. Caves form in many types of rock, either through erosion or dissolution 34 . The most common type of caves on Earth are epigenic caves, which form when meteoric water reacts with CO 2 to form a weak H 2 CO 3 solution, followed by dissolution of rock outward from the forming cave conduit and mass removal by ground water outward from the forming cave conduit 49 , 50 . Microorganisms may either accelerate or play the primary role in the formation of the second most common cave type, hypogene caves 51 , 52 . For example, hypogene speleogenesis in some sulfidic caves is driven by microbial metabolic activity within groundwater 52 , where aerobic oxidation of sulfide to sulfuric acid at or near the cave void-wall interface drives dissolution and cave formation 53 . In all cases, enlargement of the void occurs via dissolution outwards from the developing conduit 34 . In this “classical” model of cave formation, passage enlargement would be limited by intrusion of atmospheric O 2 into the cave, which would limit Fe(III) reduction. To address this paradox, we suggest a new mechanism of speleogenesis based on our results, where FeRM reductively dissolve the Fe(III)-rich matrix at the BIF/canga interface. Our previous work indicates that pulsed water flow through canga inoculated with sub muros enhances canga-Fe(III) reduction, at least partially due to removal of Fe(II) passivates, which would otherwise limit further Fe(III) reduction 31 , 33 . This pulsed water delivery, which is consistent with rainfall patterns in the rainy season, increases FeRM activity and porosity, accelerating dissolution and the formation of Fe-depleted sub muros (Fig. 8 ) 33 . Over time, this weakening of the rock matrix causes a collapse of the Fe(III)-depleted sub muros material into the cave void (Fig. 8 ). After the collapse, oxygen within the cave atmosphere auto-oxidizes the newly exposed Fe(II)-rich fluids within the still-consolidated wall structure, cementing this matrix at the cave/wall interface (Figs. 3 and 8 ) and restoring the anoxic interior of the cave wall, wherein the process repeats. The mass removal of Fe(II) by water behind the wall and resultant collapse over time creates the cave void and observed cave morphology (Fig. 1 ) 1 . The role of Fe(III) reduction and movement of water behind the walls (outside-inwards), rather than through the cave conduit (inside-outward), is a previously undescribed mechanism of speleogenesis 34 . We term this newly recognized process exothenic (from the Latin sub behind + muros wall) biospeleogenesis. Figure 8 Model for FeRM-driven dissolution and speleogenesis, consolidating the data presented here and in 1, 31, and 33 (figure adapted from 1). ( a ) The sub muros is unable to support the Fe-oxide crust on the cave wall, which collapses into the cave and enlarges the cave void. ( b ) Fe(II) exposed to oxygen in the cave passage causes auto-oxidation, which re-forms the Fe-oxide crust on the wall surface, behind which anoxic conditions then form. At the microscopic level ( b.i) under anoxia, Fe(III) serves as an electron acceptor leading to growth of FeRM, with Fe(II) production (as described in 31 ). During the dry season, the lack of flow causes Fe(II) to passivate onto the Fe-oxides, limiting Fe-reduction (as represented by an ‘x’). ( c ) During the wet season, water enters the porous canga, introducing a pulsed flow that removes passivates (as demonstrated in Ref. 33 ). These conditions ( c.i ) also bring in surface-derived organic compounds (shown as CHO) and favor Fe-reduction. ( d ) During the next dry season, the lack of pulsed flow causes the Fe(II) to begin to accumulate on surfaces again, slowing Fe-reduction and increasing FeRM abundance ( d.i ). As this cycle repeats annually, the reduction of Fe(III) increases porosity (as demonstrated in 33 ), and eventually the Fe-oxide crust becomes unstable, and the collapse and Fe-reduction cycle repeats. Over time, repeated collapse leads to wall retreat and formation of the cave passages and observed morphology. Canga is enriched in P oxides, REEs, and has a positive Eu/Eu* ratio (1.8) compared to itabirite 9 , 17 . It has been proposed that the enriched REEs in canga may be due to scavenging by secondary ferrihydrite oxides; however, this hypothesis does not take into account a role for microorganisms in this process 9 , 33 . Given the recent disruption in the global REE supply chain, there has been a renewed interest in microbial sequestration of REEs 54 . This renewed interest has helped identify a variety of mechanisms that microorganisms utilize to immobilize REEs, including biosorption and bioaccumulation, and includes member of the Chloroflexi, Proteobacteria, Acidobacteria and Actinobacteria, that are enriched in sub muros (Fig. 3 ) 31 , 33 , 54 , 55 . When we attempted to culture FeRM species from sub muros, we recovered fermentative Fe-reducing Clostridia spp. 33 . Similarly, these Clostridia appeared to use organic molecules to transfer electrons to Fe(III) with glucose as an electron donor with these lab-based FeRM cultures generating similar surface pitting profiles as those seen on hematite surfaces in situ (Fig. 4 ) 31 , 33 , 56 . Under the redox and pH conditions of sub muros, Eu would exist as Eu(III) and similar electron shuttles have been demonstrated to play a role in its reduction, which Clostridia bioaccumulate as intracellular polyphosphate 56 , 57 . Microbial activity could therefore explain the formation of canga and relative enrichment of Eu and Nd (1.8 and 4.2 compared to itabirite, respectively) 9 . In addition to identifying a new process of cave formation, these caves are an indication of the extensive potential for microbially mediated weathering of the rocks in the Carajás, Iron Quadrangle (IQ), and Southern Espinhaço Range, which are typically considered quite resistant to dissolutional weathering 1 , 9 . Recent work 12 , 13 has indicated extensive Fe cycling in canga despite the seeming permanence of the canga. The canga is continuously reworked through alternating Fe(III) reduction and then reoxidation of the biogenic Fe(II) 12 , 13 . In addition to the dynamic stability of the canga duricrust, the presence of caves and their microbial origins indicate that the itabirite phases are susceptible to substantial weathering and export of material. Here, reductively dissolved Fe may be transported through extensive subsurface aquifers, which may contribute as much as four times the freshwater discharge of rivers and streams 58 , 59 . Many of these aquifers drain directly into marine environments below the surface, such as the Mediterranean Sea, which receives up to 75% its freshwater from groundwater springs that drain without detrital material that is often associated with continental runoff 60 . With a calculated dissolution value within sub muros of 7.78 cm 3 m −3 , using a density for hematite as 5.26 g cm −3 , this would mobilize 40.9 g Fe-oxides per m 3 canga year −1 61 . If this measured reductive dissolution of Fe occurred uniformly on a regional scale, assuming the canga layer is 3 m thick, this is equivalent to ~ 122 tons of Fe annually moving into the subsurface per km 2 . Given that the Iron Quadrangle alone constitutes 7200 km 2 , even if the efficiency of this system were 1% of the in situ observed values, this is equivalent to ~ 9 million tons of subsurface Fe in this region annually. As such, itabirite weathering could be an underappreciated contributor to marine Fe budgets 62 , 63 . Indeed, continental Fe is proposed to be a substantial contributor to the high dissolved Fe in Archaean oceans from which BIF formed 65 ."
} | 4,146 |
32268518 | PMC7180454 | pmc | 4,156 | {
"abstract": "Crosslinked polymeric materials based on a quaternary trimethylammonium compound were developed and evaluated as potential antifouling coatings. For this purpose, two water-soluble random copolymers, poly(4-vinylbenzyltrimethylammonium chloride-co-acrylic acid) P(VBCTMAM-co-AAx) and poly(N,N-dimethylacrylamide-co-glycidylmethacrylate) P(DMAm-co-GMAx), were synthesized via free radical polymerization. A water based approach for the synthesis of P(VBCTMAM-co-AAx) copolymer was used. Coatings of the complementary reactive copolymers in different compositions were obtained by curing at 120 °C for one day and were used to coat aquaculture nets. These nets were evaluated in respect to their release rate using Total Organic Carbon (TOC) and Total Nitrogen (TN) measurements. Finally, the antifouling efficacy of these newly-composed durable coatings was investigated for 14 days in accelerated conditions. The results showed that this novel polymeric material provides contact-killing antifouling activity for a short time period, whereas it functions efficiently in biofouling removal after high-pressure cleaning.",
"conclusion": "4. Conclusions This work demonstrates a simple and scalable fabrication approach for the development of hydrophilic, positively charged stable surfaces from water-soluble polymers as potential antifouling coatings for aquaculture nets. Stable coatings were developed after optimization of the blend composition and the treatment procedure. The release rate of the coated nets was studied in selected simulants and showed that the fraction of the releasable material is low, about 10% ( w/w) of the whole material, suggesting that the two complementary copolymers were crosslinked to a large extent after the curing procedure. An evaluation of the antifouling activity of the coated nets was performed in tanks with seawater-nutrient solutions under accelerated conditions for a period of 14 days with a renewal of the seawater solution between the two weeks. The coated net in contrast with the uncoated one exhibited high resistance to biofouling adhesion during the first period, though after the renewal of seawater in the tank, algae settlement on the coated net was observed. However, when cleaned with high-pressure water, the fouling was removed more easily and efficiently from the coated net rather than the uncoated one. In conclusion, this cationic coating appears to provide a dual function: it acts as a biocidal against marine microorganisms for a specific time period as well as a protective surface against the biofilm adhesion on the nets. Such knowledge is a prerequisite in order to optimize the efficacy and duration of the antifouling action of this novel polymeric coating’s potential for aquaculture applications.",
"introduction": "1. Introduction The development of biofouling in marine aquaculture applications represents a crucial financial and ecological problem. Any object immersed in the sea is rapidly colonized by a wide variety of organisms [ 1 , 2 ]. These bioaccumulations have adverse effects on surfaces submerged in the aquatic environment such as shipping vessels, aquaculture cages, offshore rigs and jetties [ 3 ]. In the case of aquaculture industry, biofouling has significant impacts on culture species, on farm infrastructure (immersed structures such as cages and netting) as well as on the natural ecosystem [ 4 ]. In order to combat this problem, the aquaculture industry needs to develop optimized biofouling management technologies. An aquaculture farm usually consists of big cages which are netted with various synthetic materials. The development of biofouling on the nets, which blocks the mesh openings, is treated with periodical in situ cleaning of the fishing nets with high-pressure washers [ 5 ]. Nevertheless, this method is not cost effective, so a preventative approach is required that relies on using antifouling materials as coatings for the nets [ 6 ]. In the last few decades, the most common antifouling coatings were based on biocide-releasing paints of tributyl tin (TBT), copper and zinc-containing systems. Although very effective, these compounds raise serious issues to marine ecosystems, affecting both target and non-target organisms. For this reason, the attention of research today is focused on the development of nontoxic metal-free polymeric antifouling (AF) and fouling release (FR) surfaces. The difference between the two types of coatings is that the first one prevents the attachment of biofoulants, whereas the latter weakens the adhesion of biofoulants to the surface, facilitating their removal by hydrodynamic stress during movement or mechanical cleaning [ 7 ]. AF coatings are usually prepared from hydrophilic polymers which have high-surface energies similar to water. Therefore, they perform well as AF agents because they prefer to remain in contact with water rather than an amphiphilic biomolecule like a protein. In contrast, FR coatings are mainly composed from very hydrophobic polymers with low energy that reduces the ability of biomolecules to interact strongly with the surface. The most widely used materials for FR coatings are based on polydimethylsiloxane (PDMS) and fluorinated materials [ 8 , 9 , 10 , 11 ]. However, due to the fact that these coatings suffer from several disadvantages, they are usually combined with hydrophilic components leading to amphiphilic surfaces with improved performance [ 12 , 13 ]. Several categories of hydrophilic surfaces are being explored for the design of successful AF coatings in marine environments. Hydrogels, polymeric materials that absorb a large amount of water, have been synthesized for antifouling purpose [ 14 ]. Due to their poor stability though, recent research is focused on charged networks, which offer a promising alternative for AF coatings. Among potential candidates, zwitterionic systems appear to be highly effective against marine fouling organisms [ 15 , 16 , 17 ], as well as anionic units [ 18 , 19 ] and cationic units based on quaternary ammonium compounds (QACs) with short alkyl chains [ 20 , 21 , 22 , 23 , 24 ] which are charged compounds that although less explored in the AF field showed encouraging results. In our previous work, the reactive blending of copolymers with complementary reactive groups (AA and GMA) was applied to obtain self-standing antimicrobial membranes. Specifically, two series of copolymers poly (4-vinyl benzyl dimethylhexadecylammonium chloride-co-acrylic acid) P(VBCHAM-co-AAx) and poly (cetyltrimethylammonium 4-styrenesulfonate-co-glycidyl methacrylate) P(SSAmC 16 -co-GMAx) were combined in order to prepare crosslinked membranes, containing a quaternary N,N-dimethylhexadecylammonium group both covalently (VBCHAM) and electrostatically (SSAmC 16 ) attached [ 25 ]. These membranes presented strong antimicrobial activity against S. aureus and P. Aeruginosa, while when applied as coatings on aquaculture nets exhibited high antifouling action as compared to the blank net, for a period of up to 35 days. This methodology was investigated in more detail in order to get a deeper understanding of the release behavior of these systems [ 9 ]. An interesting observation in both works was that the release rate in salt solution was maintained in much lower levels than in pure water, offering an additional advantage for potential antifouling applications in sea water. Motivated by the above encouraging results, our aim in the present work is to investigate the role of chain length of the cationic group on antifouling activity by introducing trimethylamine instead of N,N-dimethylhexadecylamine into the P(VBC-co-AAx) copolymer. We demonstrated the quaternization reaction in a single green step using water as solvent. Taking advantage of the previously-mentioned crosslinking reaction between the acrylic acid and epoxide group, we prepared polymeric coatings of new water-soluble complementary copolymers P(VBCTMAM-co-AAx) and P(DMAm-co-GMAx). These coatings were studied for their antifouling efficiency on aquaculture nets in accelerated conditions. Thus, this study uses a simple and scalable fabrication approach for the development of hydrophilic, positively charged stable surfaces from water-soluble polymers as potential antifouling or fouling release paints.",
"discussion": "2. Results and Discussion The main idea of this work was to combine two water-soluble copolymers bearing complementary reactive groups together with positively charged ammonium groups in order to develop hydrophilic coatings stable in seawater. These materials will be further discussed in terms of their stability through release studies and of their antifouling activity in accelerated conditions. 2.1. Synthesis and Characterization of the P(VBCTMAM-co-AA20) Copolymer Precursor copolymers were synthesized via conventional free radical polymerization using AIBN as initiator ( Scheme 1 ). The structure of the synthesized copolymers was confirmed by Proton Nuclear Magnetic Resonance ( 1 H NMR) spectroscopy. The chemical composition of the synthesized copolymers was 80/20 w / w , based on the integral ratio of the broad peaks in the range 1.1–2.4 ppm which are assigned to backbone protons of VBC and AA units and the peak at 4.5 ppm attributed to the protons of chloromethyl groups of VBC. Subsequently, the introduction of trimethylamine into the benzyl chloride moiety happened in a facile route, using water as solvent. Firstly, the P(VBC-co-AA20) copolymer was dispersed in water and then the aqueous trimethylamine solution 45% ( w / w ) was added to the suspension and left to react at room temperature for one day. Finally, a homogenous opaque yellowish solution of the polymer was obtained as an evidence of the successful quaternization reaction. To the best of our knowledge, this is the first report of this reaction in water solution, while in other studies several organic solvents were used for the amination of PVBC with trimethylamine [ 26 ]. The N-quaternization reaction was verified via comparison of the 1 H NMR spectra of the product and its precursor copolymer. In particular, in the 1 H NMR spectrum of the quaternized copolymer ( Figure 1 ), it is evident that the peak located at 4.5 ppm corresponding to the protons of CH 2 Cl groups disappeared, while a new one emerges at 4.3 ppm. This new peak is attributed to the CH 2 protons (g) attached to the quaternary nitrogen of trimethylamine. In addition, the peaks at 6.7–7.2 ppm result from the aromatic protons (e,e’,f,f’) and the broad peaks at 0.8–1.8 ppm are assigned to the backbone protons of the copolymer (a,b,c,d). Finally, the peak at 2.9 ppm corresponds to the three methyl groups of the quaternary ammonium moiety. The acidic protons of the acrylic acid units do not appear in the spectrum and this is partially attributed to the formation of the trimethylammonium salt with the excess of trimethylamine and also to the exchange of the acidic protons since they do not appear in the case of the precursor copolymer P(VBC-co-AA20) neither. 2.2. Synthesis and Characterization of Copolymer P(DMAm-co-GMA30) The synthesis of the P(DMAm-co-GMAx) copolymer ( Scheme 1 ) was conducted through free radical polymerization, as reported elsewhere [ 27 ]. The chemical composition of the synthesized copolymer was estimated through 1 H NMR spectroscopy. Figure 2 shows the 1 H NMR spectrum of the P(DMAm-co-GMAx) copolymer in D 2 O with 30 mol% GMA content. Concerning GMA, the peak at 2.9 ppm corresponds to the methylene protons (h) of the epoxy ring, whereas the peak at 3.2 ppm and the broad peaks at 3.8 and 4.4 ppm correspond to the methine proton (g) of the epoxy group and the -OCH 2 protons (f) of GMA units, respectively. The α methyl group of GMA (d) appears at 0.9 ppm. The DMAm unit was confirmed by the broad peak at 2.7–3.2 ppm assigned to the methyl protons (e) of the amide. 2.3. Preparation of Antifouling Coatings The main goal of this study is the production of durable hydrophilic coatings for antifouling seawater applications. Taking advantage of our previous knowledge on the cross-linking reaction of the carboxyl group with the epoxy group after heat treatment at 120 °C [ 9 , 25 ], the two complementary copolymers P(VBCTMAM-co-AA20)/P(DMAm-co-GMA30) were blended in water and cured at 120 °C for one day in order to form crosslinked polymeric coatings ( Scheme 2 ). Since the nets that were used in this work are composed from Nylon, such conditions can be easily applied. The effect of the use of an excess of trimethylamine for the preparation of P(VBCTMAM-co-AA20) copolymer resulting in the formation of the trimethylammonium salt of the acrylic acid moieties was also investigated. Thus, copolymers synthesized with stoichiometric amount of amine were also tested and in all cases the crosslinking reaction was efficient. This was attributed to the fact that the ammonium salt of the acrylic acid reacted with the epoxides [ 28 ]. In an attempt to optimize the stability of the coatings, different compositions of the two complementary copolymers were investigated, while the content of the reactive units AA and GMA remained 20 and 30 mol%, respectively. The compositions were suitably chosen so that the obtained mixtures were rich in ammonium compounds, which were expected to provide the antifouling activity. Specifically, the content of the P(VBCTMAM-co-AA20) copolymer in the mixtures was 60%, 70% and 80% w / w . The first evidence on the success of the crosslinking reaction was obtained from the Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) characterization of membranes that were prepared by solution casting of the polymeric blends. As observed in Figure 3 for the blend P(VBCTMAM-co-AA20)/P(DMAm-co-GMA30) 80/20 w / w , the peak at 904 cm −1 which was attributed to the epoxy ring of GMA unit completely disappears after curing at 120 °C for one day. 2.4. Release Study of the Coatings The behavior of the coated nets in aqueous environment was investigated, prior to the fouling tests under accelerated fouling conditions [ 29 ]. Small pieces of the coated nets were immersed in pure water or 3.5%( w / v ) NaCl solutions and the evolution of Total Organic Carbon (TOC) and Total Nitrogen (TN) was monitored with immersion time. Indicative results concerning the behavior in salt solutions of the coated nets cured at 120 °C are shown in Figure 4 a,b. It is evident that the composition of the copolymers mixture used for coating hardly affects the behavior observed. In fact, for all three compositions, TOC and TN increase within the first few hours and then remain rather constant. These results suggest that a fraction of the polymeric material, consisted of either uncrosslinked copolymer chains or loosely crosslinked/grafted structures, is released under these conditions in the surrounding aqueous solution. The expected TOC and TN values if all material could be dissolved are indicated by the shadowed areas in Figure 4 a,b respectively. It is clear that the fraction of the releasable material is low, about 10% of the whole material, suggesting that the two complementary copolymers were crosslinked to a large extent after the curing procedure at 120 °C. In contrast, we have verified that about half of the polymeric material is released when uncured coated nets are tested under the same conditions, indicating that the crosslinking reaction is not sufficiently effective and takes place to a much lower extent at room temperature. Finally, it should be noted that the presence of salt does not affect considerably the release behavior ( Figures S1–S6 ), as expected, since neither of the complementary copolymers consists of ionic compounds that could be released to the NaCl solution through ion-exchange. The C/N molar ratios calculated from the TOC and TN values shown in Figure 4 a,b are presented in Figure 5 . The dotted lines in this Figure represent the C/N values for P(VBCTMAM-co-AA20) (upper line) and P(DMAm-co-GMA30) (lower line), as calculated from the chemical structures and also verified from the TOC and TN values determined using aqueous solutions of the two copolymers. The shadowed area represents the C/N molar ratios calculated for the three compositions of the polymer mixtures used for coating, if they were not crosslinked and were completely dissolved in the aqueous solution. As seen in Figure 5 , most of the experimental data are very close to this area, indicating that the releasable material consists of both complementary copolymers, either as free chains or as graft structures. At the very beginning of the releasing process a tendency to higher values is observed, possibly indicating that P(VBCTMAM-co-AA20) chains are more readily dissolved initially. In any case, the most important finding of these results is that this small fraction of releasable material does contain the copolymer P(VBCTMAM-co-AA20). This is crucial since this copolymer is expected to present biocidal properties, leading possibly to a release-based antifouling action. 2.5. Antifouling Test in Accelerated Conditions The blend with the highest content of cationic trimethylammonium compound P(VBCTMAM-co-AA20)/P(DMAm-co-GMA30) (80/20 w / w ) was selected to measure its antifouling performance when treated on fishing nets. The coated net along with an uncoated (blank) net were immersed in glass tanks filled with seawater and remained for 14 days. In order to achieve accelerated conditions of algae growth, 3.5 mL of a Walne medium nutrient solution and 3.5 mL of algae aliquot were added into the seawater, while four multispectral lamps with a total of 500 lux of illuminance were placed over the tanks [ 30 ]. Concerning the algal growth cycle which lasts for six to seven days, the seawater-nutrient solution was renewed on the seventh day and the experiment was carried out for another week. Photographs of the immersed nets at the beginning of the experiment and after one week are presented in Figure 6 . As may be seen there, the uncoated net (blank) had the highest fouling compared to the coated net. More specifically, enhanced algal growth is observed on the blank net accompanied by turbidity of the seawater solution. On the other hand, in the case of the coated net, even though there was a low algal growth observed at the bottom of the tank, the net itself exhibited good resistance for the seven day period. The physicochemical parameters Salinity (S), pH, Dissolved Oxygen (DO), Turbitity (Tur) and Temperature (T) were measured for the whole period of the experiment in the tanks with the uncoated and coated nets ( Table 1 ). There was no change in the salinity from the whole period of the experiment and also the turbidity of the seawater was not changed significant in the aquarium. The pH values show a gradual increase from 8.18 to 9.88 pH units and the dissolved oxygen reached the maximum value of 16.88 mg/L. Regarding the high pH values, it is expected that they will not affect the growth and production of algae during the experiment in a negative way, since the acceptable pH range for algae growth varies from pH seven to nine with the optimum range being from 8.2 to 8.7. The low temperature value 18.7 °C was observed at the beginning of the experiment and is the ambient seawater temperature, however temperature fluctuations are related with laboratory environmental conditions. In such temperature, light- and nutrient-conditioned algae growth is facilitated, photosynthesis is apparent and oxygen is produced [ 31 , 32 ]. The controlling factors for algae growth are light, nutrient availability, temperature, pH, salinity and the optimum range for these parameters applied during this study [ 31 ]. During photosynthesis, algae assimilates inorganic carbon and water, to produce organic matter and oxygen, using light as the driving source [ 31 , 32 , 33 ]. Our data indicate oxygen production during photosynthesis ( Table 1 ), however the observed depletion of oxygen after 14 days in the reference and studied aquariums was attributed to the death phase of the algae cultures due to the production of toxic metabolites. Finally, the increase of the pH values can also be attributed to the phenomenon of photosynthesis [ 32 ]. Nevertheless, right after the renewal of the seawater-nutrient solution, the settlement of fouling organisms was observed on the coated net, whereas the algal inhibition was significantly increased on the uncoated one ( Figure 7 a,b). This behavior shows that our coating does not completely prevent the fouling formation, but mainly delays the algal growth. Concerning the amount of fouling on the nets, there was not a feasible way to determine the mass of algae attached on the nets due to the co-existence of NaCl. Despite the short-term antifouling activity of the cationic polymeric coating P(VBCTMAM-co-AA20)/P(DMAm-co-GMA30) 80/20 w / w , it is important to evaluate the strength of the fouling adhesion on the coated net. For this reason, the coated and uncoated nets were cleaned under high-pressure water flow. As can be seen in Figure 7 c,d, fouling was removed easily and more efficiently from the coated net than the uncoated one. Subsequently, these results lead to the potential use of this polymeric coating to improve the life expectancy of the aquaculture nets so that they can be used several times in the field after maintenance. It should be noted that the antifouling test of the polymeric coating P(VBCTMAM-co-AA20)/P(DMAm-co-GMA30) 80/20 w / w , as well as the cleaning of the nets were repeated once and showed the same results."
} | 5,407 |
36419905 | PMC9676218 | pmc | 4,157 | {
"abstract": "Microbial electrosynthesis (MES) enables the bioproduction of multicarbon compounds from CO 2 using electricity as the driver. Although high salinity can improve the energetic performance of bioelectrochemical systems, acetogenic processes under elevated salinity are poorly known. Here MES under 35–60 g L −1 salinity was evaluated. Acetate production in two-chamber MES systems at 35 g L −1 salinity (seawater composition) gradually decreased within 60 days, both under −1.2 V cathode potential (vs. Ag/AgCl) and −1.56 A m −2 reductive current. Carbonate precipitation on cathodes (mostly CaCO 3 ) likely declined the production through inhibiting CO 2 supply, the direct electrode contact for acetogens and H 2 production. Upon decreasing Ca 2+ and Mg 2+ levels in three-chamber reactors, acetate was stably produced over 137 days along with a low cathode apparent resistance at 1.9 ± 0.6 mΩ m 2 and an average production rate at 3.80 ± 0.21 g m −2 d −1 . Increasing the salinity step-wise from 35 to 60 g L −1 gave the most efficient acetate production at 40 g L −1 salinity with average rates of acetate production and CO 2 consumption at 4.56 ± 3.09 and 7.02 ± 4.75 g m −2 d −1 , respectively. The instantaneous coulombic efficiency for VFA averaged 55.1 ± 31.4%. Acetate production dropped at higher salinity likely due to the inhibited CO 2 dissolution and acetogenic metabolism. Acetobacterium up to 78% was enriched on cathodes as the main acetogen at 35 g L −1 . Under high-salinity selection, 96.5% Acetobacterium dominated on the cathode along with 34.0% Sphaerochaeta in catholyte. This research provides a first proof of concept that MES starting from CO 2 reduction can be achieved at elevated salinity.",
"conclusion": "4 Conclusions Carbonate precipitating on cathodes (primarily as CaCO 3 ) was the critical reason for the unstable acetate production in MES systems fed with simulated seawater, likely through inhibiting the CO 2 supply, the direct electrode contact with acetogens and H 2 production. Stable acetate production by MES at seawater salinity was achieved along with a decreased system voltage and a very low apparent resistance of cathode, and further confirmed the potential in decreasing the energy input at high current densities by using the highly saline MES. The most efficient acetate production was obtained at the salinity of 40 g L −1 , because limited CO 2 dissolution and restrained acetogen metabolism at higher salinity probably inhibited acetate production. Acetobacterium , Clostridiaceae _2_unclassified, Arcobacter , and Spirochaetaceae were likely the main acetogens at seawater salinity. But under high-salinity selection, 96.5% Acetobacterium dominating on the cathode along with 34.0% Sphaerochaeta in catholyte, were presumably to be the key acetogens.",
"introduction": "1 Introduction CO 2 can be converted into commodity chemicals and fuels by carbon capture and utilization (CCU) while also contributing to the reduction in atmospheric CO 2 level and providing an alternative to petroleum products [ 1 ]. Microbial electrosynthesis (MES) is a CCU route whereby microorganisms directly or indirectly use electrical current to produce organics such as acetate from CO 2 [ [2] , [3] , [4] , [5] ]. This process could potentially contribute to achieving a sustainable society by storing renewable energy as covalent chemical bonds based on reducing CO 2 [ 5 , 6 ]. MES using whole microorganisms as biocatalysts capable of self-generation and producing long-chain organic chemicals also shows the advantages of lower overpotential, longer operational stability, and milder operational conditions [ 9 , 10 ], compared with systems employing abiotic CO 2 reduction catalysts [ 5 , 7 , 8 ]. Acetate is the most common bioproduct generated by MES, and although low in value and difficult to separate from water, this compound can serve as a precursor for the synthesis of many multi-carbon volatile fatty acid (VFA) compounds of higher economic value, such as caproate, via chain elongation [ 9 ]. Even so, the acetate production rate and the energy conversion efficiency obtainable from MES remain too low for economically viable application [ 4 , 10 ]. Acetogens remain the key microorganisms for MES using different pathways for CO 2 reduction, such as the Wood-Ljungdahl pathway [ 11 , 12 ]. These species have been reported to use cathodes either directly (via electron uptake) [ 3 ] or indirectly (via H 2 evolution) to reduce CO 2 into organics [ 10 , 13 ]. But the latter method is increasingly applied due to higher potential rates at present [ 4 , 14 ]. The low acetate production rate from MES is thus associated with the low current density reached in the bioelectrochemical system (BES) for H 2 production. This density can be up to approximately 20 mA cm −2 but generally far below. A main reason for the low current is the high ohmic resistance resulting from the low ionic conductivity of microbial-compatible electrolytes and suboptimal spacing of electrodes or the non-ideal geometry between the membrane and microorganisms [ 4 , 15 ]. It appears therefore impossible yet to combine relevantly high current densities (i.e., high production rates) and relevantly low cell voltage (i.e., acceptable energy conversion efficiencies) [ 4 ]. Reducing the high ohmic resistance in BESs is therefore crucial to improve MES performance, especially for systems with high current densities or scaled-up structures due to the development trend for MES [ 4 , 6 , 8 ]. A possible solution is to use high-salinity electrolytes, as these reduce the ohmic resistance and thereby the applied voltage needed to drive a certain current density or can increase the current produced under a certain voltage [ 16 ], which would help to improve energy conversion efficiency or production rates in MES. Although some acetogens with high salinity tolerance have been reported [ 17 , 18 ], the knowledge in the context of MES under saline conditions is very limited. A previous study assessed the use of brine pool sediment as inoculum and a seawater-brine interface solution as the electrolyte in MES systems to enrich halophilic homoacetogens at a biocathode operating at −1.0 V vs. Ag/AgCl. However, this system showed a limited capacity for VFA production at salinity levels of 25% and 10% along with a gradual decrease in reduction current [ 19 ]. Metal precipitates on the cathode were observed, but the impact of this precipitation on VFA production in the saline MES has not been revealed [ 19 ]. Slightly higher titer was subsequently observed at a salinity level of 3.5% using the former cathode as the inoculum. Genus Marinobacter and phylum Firmicutes were found to be dominant at all salinity levels examined, but no obvious acetogen was observed. Marinobacter was assumed to consume fixed carbon in this system, thus relating to the low VFA production [ 19 ]. Another study further examined MES performance at the salinity of 3.5% using salt marshes and mangrove sediments as inocula together with synthetic red seawater as catholyte at the same cathode potential [ 20 ]. This prior work used a porous ceramic hollow tube wrapped with carbon cloth as the cathode with a large projected area of 110 cm 2 for direct CO 2 delivery and minimizing CO 2 mass transfer limitation. A higher titer was subsequently obtained, but the production rates remained low at 1.69 g m −2 d −1 for acetic acid and 0.55 g m −2 d −1 for methane after a 10-day batch operation, while the reduction current also decreased over time [ 20 ]. During these trials, Acetobacterium belonging to the phylum Firmicutes exhibited unstable enrichment, which was likely related to the low acetate production rate [ 20 ]. Cathodes in the aforementioned studies were both poised at −1.0 V vs. Ag/AgCl. Notably, due to cathodic hydrogen production, the pH in the vicinity of the cathode will increase and this could lead to scale formation by the Ca 2+ and Mg 2+ ubiquitous in seawater and other saline streams. How this will impact the electrochemical activity and hydrogen production performance under that potential is as yet not studied, and it makes unclear whether high salinity will actually improve MES. Although the microbial community regarding CO 2 reduction in cathodic chambers under high salinity has been analyzed in previous studies [ 19 , 20 ], the community effectively contributing to acetate production under high salinity is also as yet not defined. The present work investigated acetate production under saline conditions while assessing the impact and mechanism of scaling on MES. Reactors were operated stably for 137 days at the salinity of 35 g L −1 (seawater salinity), after which the salinity was increased to 60 g L −1 to study the MES performance under the elevated salinity. The microbial community composition and its contribution to acetate production in the saline MES system were analyzed based on the 16S rRNA gene and quantitative polymerase chain reaction (qPCR) analyses.",
"discussion": "3 Results and discussion 3.1 Unstable acetate production under a potentiostatic condition Acetate was produced in S-MES system with a cathode potential of −1.2 V using simulated seawater as catholyte but not unstably ( Fig. 1 ), which also occurred in previous studies [ 20 , 25 ]. Acetate along with small amounts of formate and propionate was produced in this system during three successive cycles. However, the peak acetate concentration gradually decreased for each cycle, reaching 1,407, 969, and 400 mg L −1 , respectively ( Fig. 1 a). The maximum production rate ( R max ) also decreased from 20.15 to 9.27 then to 5.44 g m −2 d −1 , accompanied by a decline in the average acetate production rate ( R avg ) from 9.28 ± 6.34 to 3.85 ± 4.09 then to 0.99 ± 2.78 g m −2 d −1 ( Fig. S2 ). A decrease in the reductive current at −1.2 V was observed during these cycles ( Fig. 1 b). The CE for cumulative VFAs in each cycle also decreased, from 49.1 ± 0.1% to 22.6 ± 0.1% ( Fig. 1 c and Fig. S2c ), suggesting that the energy conversion efficiency was lowered. In addition, although H 2 was detected prior to inoculation, the gradual decrease in the reductive current and the relatively high purge rate resulted in a minimal amount of residual H 2 at the end of each cycle after consumption by microbes. But the electron involved in residual H 2 may also contribute to the low CE. The decrease in the reductive current density was also confirmed by CV characterization (with −7.66, −7.0, and −1.43 A m −2 at −1.2 V in cycle 1, 2, and 3, respectively, Fig. 1 d). The CV results indicated an increasingly weak electrochemical activity of the cathode for H 2 production. Fig. 1 Acetate production performance of S-MES with cathode poised at −1.2 V vs Ag/AgCl using simulated seawater as catholyte. a , VFA production; b , The chronoamperogram of the cathode; c , Coulombic efficiency for cumulative VFA production; d , Cathode CV scan results. Fig. 1 A white coating was observed forming on the S-MES system cathode during these experiments. The SEM-EDX analysis revealed that those precipitates mostly contained Ca with minor amounts of Mg and P ( Fig. 2 ). The XRD analysis further indicated that the white precipitates primarily comprised CaCO 3 (JCPDS No. 84–0049) ( Fig. 2 c). These results agreed with the production of numerous bubbles during the dissolution of these precipitates with 1.00 M HCl ( Fig. S3 ) and the higher mass-based ratio of Ca 2+ to Mg 2+ in the resulting solution (1.50) compared with the original simulated seawater (0.32). NaCl was also found in this precipitate due to the use of saline catholyte (JCPDS No. 78–0751). Those insulating carbonate precipitates likely hindered the direct contact between both water (acting as a hydroxyl ions source) and microbes with the cathode surface and thus inhibited H 2 production. The direct contact between the cathode and acetogens may be also important for acetate production, because microbes have been reported to add catalysts at the cathode surface to facilitate CO 2 reduction by producing intermediates [ 15 ]. Fig. 2 a – b , SEM-EDX micrographs of the S-MES cathode after the experiment with precipitation on the electrode surface ( a ) and a new cathode before the experiment ( b ). c , The XRD patterns of the white precipitate and pure CaCO 3 and NaCl. Fig. 2 3.2 Unstable acetate production at a −10 mA reductive current To eliminate the impact of insufficient H 2 generation on acetate production, the acetate production using simulated seawater as catholyte was further studied at a reductive current density of −1.56 A m −2 in B-MES system with a constant H 2 yield. In this trial, acetate was again the main product, along with minor amounts of propionate and butyrate ( Fig. 3 a). Formate was also detected during the first two cycles, likely because of the high catholyte pH (8.43–8.85) during the second half of cycle 1 ( Fig. 3 a) [ 13 ]. It should be noted that the acetate production was again unstable and the peak acetate concentration decreased from 1287 mg L −1 in cycle 1 to 783 mg L −1 in cycle 3 ( Fig. 3 a). The R max also decreased from 8.52 in cycle 1 to 3.06 g m −2 d −1 in cycle 3 ( Figs. S4a–c ) and R avg declined from 2.99 ± 2.73 to −0.30 ± 3.92 g m −2 d −1 . The average CE for cumulative VFA production stabilized at 19.2 ± 1.5% in cycle 1 but sharply decreased to 4.5% in cycle 3 ( Fig. S4g ) along with a gradual increase in the system voltage from 2.58 to approximately 2.66 V ( Fig. S4e ). The CEs for H 2 at the end of these cycles were within 16.0–22.0%. A higher instantaneous CE for VFA production was observed in conjunction with a high acetate production rate during cycle 1, such as 61.5% on day 19 and 86.0% on day 23 ( Fig. S4g ). These data indicated that the low amount of microbes at the beginning of cycle 1 may contribute to the low CE for VFA generation. But this CE remained low during cycles 2 and 3 and even became negative during the second half of cycle 3 ( Fig. S4h ), which suggested that the reduction of CO 2 to generate VFAs was inhibited, and a portion of VFAs was likely converted to other compounds or consumed by microbes [ 19 ]. The pH at the end of each cycle stabilized at approximately 8.5, and white precipitates were again deposited on the cathode surface ( Fig. S3c ). Because the H 2 yield was constant due to the constant reductive current, the carbonate precipitates could possibly reduce both the CO 2 supply and H 2 utilization, leading to a decrease in CE related to VFA production. Fig. 3 Acetate production performance of B-MES with a reductive current density of −1.56 A m −2 using simulated seawater as catholyte during the first three cycles but lower Ca 2+ and Mg 2+ concentrations in cycle 4. a , VFA production; b , Cathode CV scan results. Fig. 3 To address the scaling issue, the hardness of the catholyte used in cycle 4 was lowered as described in Section 2.1 , following which stable acetate production was achieved. The catholyte pH remained at approximately 8.5, but the acetate concentration continually increased over the remaining 60 days, reaching a value of 5193 mg L −1 ( Fig. 3 a) along with a R avg of 3.51 ± 1.69 gm −2 d −1 . The average CE for cumulative VFA production also increased to 25.1 ± 4.4%, and the instantaneous CE at the end of the cycle was significantly higher at 58.3%, along with a low CE for residual H 2 of 5.3% ( Fig. S4g ). These results were consistent with the observed decrease in cell voltage to 2.55 V ( Fig. S4e ) and the recovery in the cathode potential from −1.42 to −1.34 V ( Fig. S4f ). Successive CV data showed obviously higher reductive current at −1.34 V indicating a more rapid H 2 removal from the cathode surface by microbes ( Fig. 3 b) [ 22 ]. The alkaline pH in the vicinity of the cathode resulting from cathodic hydrogen production likely favored carbonate formation and thus generated primarily CaCO 3 . Therefore, the carbonate precipitates further decreased CO 2 availability and also inhibited H 2 generation and its utilization by acetogens. The formation of calcium carbonate precipitates was likely the main reason for the unstable acetate production in these MES systems under saline conditions. 3.3 Stable long-term acetate production at −10 mA in a three-chambered BES The 3C-B c -MES system was constructed to further investigate stable acetate production under saline conditions while avoiding the common phenomenon of chloride oxidation at the anode [ 20 ]. In this system, the anolyte was a low-pH Na 2 SO 4 solution to enhance the proton supply to catholyte. But in the trail using synthetic seawater as catholyte and the Na 2 SO 4 solution as anolyte in this system without inoculation, tight white precipitates were again formed on the cathode surface and obviously inhibited the cathode electrochemical activity ( Fig. S5 ). Thus, the catholyte was replaced with synthetic seawater with a lower hardness, as described in Section 2.1 . Stable acetate production was obtained using this system during three batch cycles throughout 137 days ( Fig. 4 ). The cumulative acetate concentration was as high as 3440 mg L −1 within a single cycle, and the R max reached 8.99 ± 1.30 g m −2 d −1 along with a R avg of 3.80 ± 0.21 g m −2 d −1 during these three cycles. One mole of acetate production is equivalent to 2 mol of CO 2 fixation, and the biomass of chemolithoautotrophic microbes producing acetate from CO 2 and H 2 was reported to accounted for 5% of the carbon flux [ 23 , 24 ]. Therefore, the maximum CO 2 consumption rate was 13.84 ± 2.01 g m −2 d −1 and the average was 5.94 ± 0.30 g m −2 d −1 . The average CE for cumulative VFA production during these three cycles was 36.0 ± 3.0% ( Fig. 4 c), essentially entirely as acetate (34.4 ± 3.2%). The instantaneous CE for VFA generation was also higher during the second half of each cycle, with average values of 42.2 ± 19.8%, 45.7 ± 23.3%, and 40.1 ± 16.9% for cycles 1, 2, and 3, respectively ( Fig. S6 ). These data also suggested limited usage of H 2 by low amounts of microbes at the beginning of the cycle, leading to the relatively low CE for cumulative VFA. The instantaneous CE for H 2 during these cycles fluctuated within the range of 11.3–34.0%, likely due to the relatively high flow rate of the mixed gas. Methane formation was not detected, but some electrons may also have ended up in the biomass. The CV results shown in Fig. 4 d confirmed that this cathode exhibited more stable and higher electrochemical activity during long-term operation likely due to the absence of precipitation on the cathode surface when compared with the B-MES system. An oxidative peak at approximately −0.57 V (vs. Ag/AgCl) was observed, but probably did not play an important role as the actual cathode potential was much lower [ 22 ]. A stable, lower voltage of approximately 2.45 V was obtained ( Fig. 4 b) along with a higher cathode potential of approximately −1.23 V. These results could be attributed to the low energy losses in this system without the formation of precipitates on the cathode. Fig. 4 Stable acetate production performance of 3C-B c -MES at seawater salinity (35 g L −1 ) with low Ca 2+ and Mg 2+ concentrations. a , VFA production; b , System voltage; c , Coulombic efficiency based on cumulative VFA production; d , Cathode CV scan results. Fig. 4 Because these systems were purged solely with 10% CO 2 at a relatively high rate and operated at a low current density, the VFA production rate was likely slowed. Even so, this rate was still much higher than those observed in prior studies using the same salinity (0.95 and 0.46 g m −2 d −1 for acetate and formate, respectively [ 19 ], and 1.69 and 0.55 g m −2 d −1 for acetic acid and methane, all based on projected cathode area, respectively) [ 20 ]. The apparent resistance of cathode, as determined by the current interrupt method [ 26 ] showed a quite low level of 1.9 ± 0.6 mΩ m 2 for the present system ( Fig. S7 ) and thus agreed with the high catholyte conductivity of 50.1 ± 0.8 mS cm −1 at the end of the cycle ( Fig. S8 ). This resistance was much higher in B-MES and S-MES systems with 71.8 and 6.4 mΩ m 2 , respectively, likely due to precipitation on their cathode surfaces. BESs have been reported to show higher area-specific ohmic resistance, typically higher than 10 mΩ m 2 because of the low ionic conductivity of microbial compatible catholyte (generally around 10 mS cm −1 ) [ 4 , 26 ]. Considering that the electrolysis of water typically proceeds at the anode, this low apparent resistance of cathode would be expected to greatly affect the energy efficiency of MES, especially at high current densities [ 4 , 8 ]. The voltage used in this research was lower than values used in prior studies but was combined with a higher current density ( Table S1 ), further confirming the decreased energy input required by highly saline MES processes. In addition, because the CEM was permeable to salt [ 27 ], chloride appeared in the anolyte of this system. But significantly more chloride was accumulated in the middle chamber and thus subsequently prevented severe chloride oxidation on the anode ( Fig. S9 ). 3.4 Acetate production with elevated salinity at −10 mA Building on the stable performance of MES in 3C-B c -MES system, another three-chamber MES system (designated 3C–B i -MES) was constructed to investigate acetate production at increasingly elevated salinity levels between 35 and 60 g L −1 . The catholyte conductivity at each salinity gradually increased from 48.7 to 78.1 mS cm −1 ( Fig. S8 ), which was consistent with the salinity settings. Acetate was continually produced when the salinity was gradually increased from 35 to 50 g L −1 , resulting in significant accumulation of this product ( Fig. 5 a). However, R max gradually decreased to 8.94, 8.70, 5.47, and 4.21 g m −2 d −1 at salinity levels of 35, 40, 45, and 50 g L −1 , respectively, while R avg stabilized at 3.93 ± 2.01, 4.56 ± 3.09, 3.24 ± 1.51, and 3.31 ± 0.71 g m −2 d −1 under each salinity ( Fig. S10 ). The maximum CO 2 consumption rates at these same salinity levels were 13.77, 13.40, 8.42, and 6.48 g m −2 d −1 , respectively, while the average values were 6.05 ± 3.10, 7.02 ± 4.75, 4.50 ± 2.32, and 5.10 ± 1.10 g m −2 d −1 , respectivley [ 23 , 24 ]. The average CEs for cumulative VFA production were 32.2 ± 3.0%, 38.6 ± 3.3%, 35.6 ± 5.4%, and 30.4 ± 1.1%, respectively, at each salinity ( Fig. 5 c). The instantaneous CEs for VFA production were again higher than the CE for cumulative VFA production, especially at salinity levels of 35 and 40 g L −1 with average values of 43.4 ± 22.5% and 55.1 ± 31.4%, respectively ( Fig. S10d ), which also suggested limited uptake of H 2 by the low amounts of microbes at the beginning of the cycle. The instantaneous CE for VFA production at 45 and 50 g L −1 were substantially reduced to 24.7 ± 16.0% and 32.4 ± 7.5%, respectively, along with the decreases in the instantaneous CEs for H 2 generation at the end of the operating period from 5.3% at 35 g L −1 to 2.6% at 50 g L −1 . These results indicated the inhibition of CO 2 reduction to VFAs at higher salinity. When the salinity was further increased to 60 g L −1 , acetate production was inhibited in the first eight days and then recovered in the following days, but the average production rate and CE were very low ( Fig. 5 c and Fig. S10d ) which could possibly be attributed to adaption of the acetogens. Fig. 5 Acetate production performance of 3C–B i -MES as the salinity gradually increased from 35 to 60 g L −1 in conjunction with low Ca 2+ and Mg 2+ concentrations. a , VFA production; b , System voltage; c , Coulombic efficiency for cumulative VFA production; d , Cathode CV scan results. Fig. 5 Notably, the system voltage also gradually declined from 2.45 to 2.29 V ( Fig. 5 b), while the cathode potential gradually rose from −1.19 to −1.07 V ( Fig. S10e ) upon raising the salinity from 35 to 40 g L −1 and remained at this level as the salinity increased above 40 g L −1 . The apparent resistance of cathode also gradually decreased from 1.9 mΩ m 2 at 35 g L −1 to 1.2 mΩ m 2 at 60 g L −1 ( Fig. S7 ). The slight decrease in the system voltage could be attributed to the very low apparent resistance at these salinity levels. The CV data demonstrated that the cathode activity was significantly improved as the salinity was increased ( Fig. 5 d). Specially, H 2 evolution potential was −0.97 V at 35 g L −1 after inoculation but increased to −0.90 V at salinity levles within the range of 45–60 g L −1 ( Fig. 5 d). The extent to which CO 2 dissolves has been reported to decrease at high salinity, which would, in turn, be expected to lower the production of VFAs [ 19 , 20 , 28 ]. Thus, the highest CE appeared at 40 g L −1 likely due to the decreased cell voltage and a presumably slight decrease in CO 2 solubility at this salinity. The inhibited VFA production at higher salinity may also relate to the energy conservation because microbes may produce organic compatible solutes such as ectoine due to osmotic adaption [ 19 , 29 ]. In addition, when the salinity was no less than 45 g L −1 , a redox couple appeared during CV scan with an oxidative peak at approximately −0.71 V and reductive peak at −1.08 V. The half-saturation potential ( E M ) of this redox couple was −0.90 V, which was similar to the H 2 evolution potential at high salinity. However, this redox couple was unstable as these peaks became increasingly weak with continued CV scans ( Fig. S11 ). But the peaks returned following an extended period of polarization, which was in agreement with the previous research in which an oxidative peak corresponding to H 2 oxidation was significantly enhanced after a prolonged polarization period when using a microbes-covered graphite electrode [ 30 ]. This phenomenon may be related to enzyme activation processes induced by extended electrode polarization [ 30 ]. Therefore, this couple may be associated with the shift in cathode potential and the significantly increased reductive current at high salinity. As no similar reductive peak was observed in the present MES systems at the salinity of 35 g L −1 , so this redox couple might represent a unique feature of the microbial communities that developed at salinity levels higher than 35 g L −1 , as discussed below (Section 3 . 5.2). 3.5 Microbial community analysis The SEM images of cathodes used in these systems ( Fig. 6 ) show that biofilms were successfully formed on the cathode surfaces in the three-chambered systems of 3C-B c - and 3C–B i -MES with low-hardness catholyte. Microbes were also found on cathodes from both S- and B-MES systems but primarily on the surfaces of thick CaCO 3 precipitates that deposited on the cathode surfaces. Those outcomes concurred with the inhibited cathode electrochemical activity and acetate production in those two systems. Fig. 6 SEM images of biofilms on cathodes from different systems and of the blank cathode. a , S-MES; b , B-MES; c , 3C-B c -MES; d , 3C–B i -MES systems; e , The blank cathode. Fig. 6 3.5.1 The effects of scaling ions on MES microbial activity Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla in B-MES system but with obviously higher relative abundances of the first two phyla (together accounting for 85–91%). Firmicutes were highly enriched on the cathode (75.8%), but more Proteobacteria were accumulated in catholyte (58.7%) ( Fig. S12 ). These two phyla were also accumulated in 3C-B c -MES system with the same trends. Firmicutes, Proteobacteria, and Actinobacteria have been reported to contain many CO 2 -assimilating microorganisms [ 11 ]. Additionally, minimal Epsilonbacteraeota were enriched in all catholyte, while Spirochaetes were only obviously enriched in the 3C-B c -catholyte. In genus level, the most abundant acetogen accumulating on the cathode in both B-MES and 3C-B c -MES systems was the canonical homoacetogen Acetobacterium [ 31 ], with relative abundances of 67.1% and 78%, respectively ( Fig. 7 ). This genus was very likely the primary carbon fixer reducing CO 2 to acetate via the Wood-Ljungdahl pathway, and probably did a significant contribution to VFA production in these systems [ 15 ]. This genus was also found in catholyte but at much lower levels (19.8% in B–P and 3.8% in 3C-B c -P). Such results concurred with previous studies, which suggested that the cathode and biocatalyst should be in close proximity to one another to facilitate the transfer of electron mediators such as H 2 during CO 2 reduction [ 6 , 15 ]. Therefore, the direct contact with the cathode was evidently important for acetate formation. The carbonate precipitates likely restricted this direct contact between the acetogens and the cathode and also limited the CO 2 supply, leading to weakened cathode electrochemical activity. The proportion of Acetobacterium in 3C-B c -P catholyte samples also decreased from 19.0% to 3.8% during the batch cycles. This decrease could probably be attributed to the lower Ca 2+ and Mg 2+ levels in catholyte that, in turn, led to better contact with the electrode. As such, the metabolism of this dominant genus on the cathode was promoted, and the H 2 concentration in the catholyte was decreased. Some other acetate-producing genera were also accumulated in these systems but with much lower proportions, within the range of 4.4–8.8%, including Clostridiaceae _2_unclassified, Propionibacteriaceae _unclassified, and Sphaerochaeta . Fig. 7 Microbial community composition of the cathode and catholyte consortia from B-MES, 3C-B c -MES, and 3C–B i -MES systems in genus level over 1% based on 16S rRNA gene analysis. Fig. 7 Besides, Pseudomonas was the most abundant genus in catholyte with 56.7% in B–P and 68.4–71.9% in 3C-B c -P, but with obviously lower proportions on the cathode (11.6–13.6%) ( Fig. 7 ). This genus has been reported to be highly enriched both on the cathode and in catholyte of MES systems for industrial CO 2 reduction (up to 26% relative abundance) and was thought to promote extracellular electron transfer and hydrocarbon removal [ [31] , [32] , [33] ]. The exoelectrogenic genus Arcobacter present in the catholyte of 3C-B c -P with 7.0% might associate with the enrichment of Pseudomonas producing electron mediators. The significant proportion of Pseudomonas in the catholyte may also affect both H 2 oxidation and acetate consumption in the system, because of its hydrogenase activity and fermentation capacity [ 33 , 34 ], but further study is required to definitively assess the function of this genus in such systems. Finally, the fermentative genus Bacteroidia _unclassified was gradually accumulated in 3C-B c -P with relative abundance of 7.0%, 6.3%, and 11.2% at the end of cycles 1, 2, and 3, respectively. These microbes' presence and activity might help explain the low acetate production rate at the beginning of the cycle. 3.5.2 Microbes contributing to efficient VFA production at high salinity In the 3C–B i -MES system, only Firmicutes (96.6%) and Proteobacteria (1.5%) were accumulated on the cathode at 60 g L −1 salinity likely due to the high salinity tolerance and unique ability of these phyla to acquire energy on the cathode under highly saline conditions ( Fig. S12 ). The dominant phyla in catholyte at this salinity mainly contained Proteobacteria (57.0%) and Spirochaetaceae (34.0%). Acetobacterium was essentially the only genus on the cathode, with a proportion of 96.5%. However, the proportion of this genus in catholyte was much lower, which increased from an initial value of 2.3% to 11.0% as the salinity was gradually increased to 45 g L −1 but then decreased to 1.1% at 50 g L −1 and 0.4% at 60 g L −1 . These results could be related to low CO 2 solubility [ 35 ], a reduced H 2 concentration in the catholyte, and a possibly inhibited metabolism of this microbe at high salinity. The proportions of the other acetate-producing genera, including Arcobacter , Propionibacteriaceae _unclassified, and Clostridiaceae spp., were also significantly decreased to very low levels (below 2%), likely also due to the limited tolerance of these microbes to the high salinity. These lower amounts of these genera probably also related to the decrease in acetate production under hypersaline conditions. Nevertheless, certain genera tolerable to high salinity were gradually enriched in catholyte as the salinity was increased, such as Sphaerochaeta which went from an initial proportion of 1.7% to 17.3% at 45 g L −1 and then to 33.5% and 34.0% at 50 and 60 g L −1 , respectively. This accumulation could be attributed to the halophilic nature of this genus and its capacity for oxidative acetogenesis under highly saline conditions [ 32 , 36 ]. The generation of H 2 and CO 2 by this genus may benefit the metabolisms of other acetogens in catholyte at high salinity. Halomonas and Shewanella tolerant of high salinity were both also gradually accumulated in catholyte at salinity levels of 50 and 60 g L −1 within 2.5–1.5%. Meanwhile, Pseudomonas remained the most abundant genus in catholyte, decreasing from an initial abundance of 77% to 58.6% at 45 g L −1 , and 48.5% and 51.3% at 50 and 60 g L −1 , respectively. But the proportion of this genus on the cathode was only 1.4%, suggesting low electrochemical activity at high salinity. In addition, Bacteroides was also accumulated only in catholyte to proportions of 1.7–4.9% with salinity increases, likely due to the low acetate production rate. According to the qPCR analysis shown in Fig. S13 , the absolute abundance of Acetobacterium was 3.46 × 10 11 copies on the cathode, which was 238 times higher than that in catholyte (with an initial value of 9.22 × 10 8 followed by an increase to 1.00 × 10 10 at 45 g L −1 but decreases to 3.38 × 10 9 and 1.46 × 10 9 at salinity levels of 50 and 60 g L −1 , respectively). Furthermore, even taking all the copies of acetogens in catholyte into account, including Arcobacter , Propionibacteriaceae _unclassified, Clostridiaceae _2_unclassified, and Sphaerochaeta , the total amount was still much lower (with copies of 1.25 × 10 11 , 1.18 × 10 11 and 1.47 × 10 11 at 45, 50, and 60 g L −1 ). Thus, the Acetobacterium on the cathode likely played a significant role in VFA production within this highly saline system. But the change in the absolute abundance in catholyte provided further evidence that this genus exhibits a suitable metabolism at salinity below 45 g L −1 but a highly inhibited metabolism at 50 and 60 g L −1 . In combination with the special redox couple observed during the cathode CV scans at salinity above 40 g L −1 , the pronounced accumulation of this genus on the cathode might indicate a specific extracellular electron transfer pathway in Acetobacterium that supports its metabolism along with inhibited VFA production, but a further study is required. In addition, the special enrichment of Sphaerochaeta in the catholyte (with an increase from the initial copies of 6.7 × 10 8 to 1.58 × 10 10 at 45 g L −1 and to 1.01 × 10 11 and 1.27 × 10 11 at 50 and 60 g L −1 , respectively) might also relate to the redox couple appearing during the CV scans at salinity levels no less than 45 g L −1 . Overall, Acetobacterium was evidently the key acetogen for VFA production in the present MES system under hypersaline conditions and primarily accumulated on the cathode. The special accumulation of Sphaerochaeta in the catholyte may also contribute to acetate production. The lower acetate production rates observed at salinity levels higher than 40 g L −1 presumably resulted from the limited CO 2 solubility and the partial inhibition of acetogenic metabolism at high salinity."
} | 9,013 |
32140381 | PMC7050239 | pmc | 4,159 | {
"abstract": "Abstract Camouflage and wound healing are two vital functions for cephalopods to survive from dangerous ocean risks. Inspired by these dual functions, herein, we report a new type of healable mechanochromic (HMC) material. The bifunctional HMC material consists of two tightly bonded layers. One layer is composed of polyvinyl alcohol (PVA) and titanium dioxide (TiO 2 ) for shielding. Another layer contains supramolecular hydrogen bonding polymers and fluorochromes for healing. The as‐synthesized HMC material exhibits a tunable and reversible mechanochromic function due to the strain‐induced surface structure of composite film. The mechanochromic function can be further restored after damage because of the incorporated healable polyurethane. The healing efficiency of the damaged HMC materials can even reach 98 % at 60 °C for 6 h. The bioinspired HMC material is expected to have potential applications in the information encryption and flexible displays."
} | 241 |
39580490 | PMC11585574 | pmc | 4,160 | {
"abstract": "Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display metabolic and transcriptional diversity along with recalcitrance to antibiotics and host immune defenses. Here, we present an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. BaSSSh-seq captures extensive transcriptional heterogeneity during biofilm compared to planktonic growth. We quantify and visualize transcriptional regulatory networks across heterogeneous biofilm subpopulations and identify gene sets that are associated with a trajectory from planktonic to biofilm growth. BaSSSh-seq also detects alterations in biofilm metabolism, stress response, and virulence induced by distinct immune cell populations. This work facilitates the exploration of biofilm dynamics at single-cell resolution, unlocking the potential for identifying biofilm adaptations to environmental signals and immune pressure.",
"introduction": "Introduction Bacterial infections represent a pervasive clinical problem that is increasingly complicated by the emergence of multidrug-resistant (MDR) strains, recognized as one of the greatest threats to human health worldwide 1 – 4 . One successful bacterial pathogen typified by MDR is Staphylococcus aureus ( S. aureus ) 5 . While a commensal in nearly one-third of the human population, S. aureus is transmitted across both hospital and community settings as a leading cause of post-surgical infection, skin and soft tissue infection, bacteremia, endocarditis, osteomyelitis, and medical device-associated infection 6 . In addition to the large arsenal of immune evasion molecules and antibiotic resistance genes encoded by S. aureus , a hallmark of this pathogen is its propensity for biofilm formation 6 , 7 . Biofilm is a key mechanism for survival and persistence in the infected host, leading to significant morbidity and mortality not only for S. aureus , but also other MDR pathogens including Escherichia coli , Klebsiella pneumonia , and Pseudomonas aeruginosa 8 . It has been estimated that approximately 65% of nosocomial infections are associated with biofilm formation 9 . Encased in an extracellular matrix comprised of polysaccharides, proteins, and nucleic acids, the multicellular biofilm community is highly recalcitrant to antibiotics and the host immune system 7 – 9 . A combination of bulk transcriptomics, bacterial mutants, and fluorescent reporter strains have been employed to identify metabolically and transcriptionally diverse subpopulations of bacterial cells within biofilm that have differing roles in surface attachment, dispersal and dissemination, stress-response, host defense, and persistence 7 , 8 . Understanding these communities has been hindered by lack of a high-throughput method to simultaneously measure the complex and stochastic interactions between distinct bacterial subpopulations. Single-cell RNA sequencing (scRNA-seq) is widely used for transcriptional profiling of eukaryotic cells within a heterogeneous sample 10 . It has been applied to assess immune response dynamics during bacterial infection, including biofilm, identifying transcriptional changes in leukocyte metabolism, reactive oxygen species (ROS) production, and inflammatory mediator signaling specific to each immune cell type 11 – 15 . However, the use of scRNA-seq has traditionally been limited in prokaryotes based on the short half-life and low abundance of mRNA, lack of polyadenylated transcripts, and complex cell wall characteristics 16 – 18 . As a result, bulk RNA-seq methods have primarily been used to study bacterial pathogens and biofilm communities. However, bulk methods fail to capture heterogeneity and underrepresented populations altogether. A single-cell approach is necessary for a complete transcriptional landscape of biofilm heterogeneity and how biofilm is affected in response to distinct immune pressures, a critical step towards identifying novel anti-biofilm strategies. Only recently have bacterial scRNA-seq methodologies been described, each employing unique protocol variations with respective pros and cons 19 – 27 . One major area of variation between described methods is how individual cells are labeled with distinct oligonucleotide barcode sequences for identification, with methods broadly separating into plate- and microfluidics-based barcoding approaches. Plate-based systems have utilized standard 96- or 384-well plates that impose inherent limitations on cell numbers 19 , 21 , 24 , while microfluidics-based approaches permit acquisition of increased cell numbers but require adaptation of costly commercial instrumentation 22 , 23 , 25 , 26 . Another technique employed fluorescence-activated cell sorting for bacterial cell separation and identification, but yields were limited to a few hundred cells 20 , 27 . A second major area of methodological variation is RNA capture, with most approaches utilizing random hybridization or mRNA-targeted probes. The use of targeted mRNA probes requires prior knowledge of the genome and desired targets, effectively limiting the number of genes analyzed 23 , while random hybridization provides unbiased insights into all possible genes but results in an overabundance of rRNA reads (i.e. >90%) 19 , 21 , 22 , 24 , 25 . Initial studies with random RNA hybridization omitted rRNA depletion, whereas more recent reports successfully incorporated rRNA depletion with Cas9 or RNase H 22 , 25 , 26 . In all published bacterial scRNA-seq methods to date, studies were limited to planktonic organisms and focused on proof-of-concept feasibility. Several reports examined transcriptional changes between different planktonic growth states 19 , 21 , 26 , whereas others observed transcriptional variation in planktonic culture upon treatment with antibiotics or other stimuli 22 , 23 , 25 , 26 . Here, we present an advanced method and application of bacterial scRNA-seq to explore the heterogeneity of complex biofilm communities and transcriptional adaptations in response to immune cell challenge. Our technique, termed BaSSSh-seq ( ba cterial scRNA-seq with s plit-pool barcoding, s econd strand synthesis, and s ubtractive h ybridization), employs an optimized protocol for RNA capture from bacterial cells with low metabolic activity, as seen in biofilm 7 , 8 . BaSSSh-seq uses plate-based split-pool barcoding to label individual cells, without the need for sophisticated commercial equipment 28 – 31 . Random hexamers are used for unbiased RNA capture during barcoding. Additionally, second strand synthesis replaces the highly inefficient process of template switching to generate cDNA libraries 32 , and an enzyme-free rRNA depletion method based on subtractive hybridization is used to significantly reduce rRNA contamination 33 . Through reduced enzyme usage and rRNA contamination, costs are decreased while concurrently increasing sequencing depth. This concept is important for bacterial scRNA-seq given the inherent sparseness of cellular mRNA. We established that diversity can be captured from bacterial cells with low metabolic and transcriptional activity within biofilm and coupled this with innovative computational assessments for identifying transcriptional heterogeneity and dynamics. We applied BaSSSh-seq to study unique transcriptional signatures that differentiate S. aureus biofilm from planktonic growth and how biofilm alters its transcriptional profile in response to immune pressure, elevating bacterial scRNA-seq from proof-of-concept demonstrations to address complex biological interactions. An initial comparison of biofilm vs. planktonic growth demonstrated the ability to capture transcriptional heterogeneity within biofilm and validated the BaSSSh-seq methodology through extensive consistency with literature and experimental observations. We then explored biofilm transcriptional alterations in response to immune pressure by applying BaSSSh-seq to biofilm after direct co-culture with three major leukocyte populations that have well-documented roles in S. aureus infection: macrophages (MΦs), neutrophils (PMNs), and granulocytic myeloid-derived suppressor cells (G-MDSCs) 34 – 36 . Within the transcriptionally diverse subpopulations of biofilm, differential responses to each leukocyte population were observed. We further developed an analytical pipeline using a combination of unique computational assessments and existing bioinformatics packages for an enhanced multi-level visualization of biofilm transcription. Through integration of iModulon analyses, we achieved a high-level assessment of transcriptional regulatory networks across biofilm subpopulations in addition to gene-level characterization 37 – 39 . Likewise, trajectory analysis was used to identify transcriptional dynamics between S. aureus growth states and activation upon immune pressure 40 . Together, BaSSSh-seq provides the opportunity for studying biofilm growth dynamics and interactions with the immune system at a new level of resolution, promoting enhanced understanding of biofilm pathogenesis and the potential for rational design of new therapeutic strategies.",
"discussion": "Discussion Here we present BaSSSh-seq, a bacterial scRNA-seq method incorporating a plate-based barcoding system with rRNA depletion. BaSSSh-seq was applied to study S. aureus biofilm heterogeneity and immune interactions, an advance from previous demonstrations of bacterial scRNA-seq on planktonic cells. This application captured vast transcriptional heterogeneity within biofilm compared to planktonic growth and permitted the detection of distinct biofilm responses tailored to different immune cell populations. In addition to the technical advances in scRNA-seq methodology, our analyses present a conceptual advance toward the understanding of complex biofilm communities by incorporating new computational pipelines that enable high-level regulatory network visualization and trajectory inference paired with gene-level expression quantification. Our BaSSSh-seq methodology was validated by literature comparing alterations in gene expression and metabolism during biofilm vs. planktonic growth. Moreover, subsequent analyses laid the groundwork for exploration beyond simple validation. A current lack of understanding exists surrounding the intricately coordinated cellular networks that govern biofilm growth, stemming from inadequate high-throughput methods to measure the stochastic interactions between discrete subpopulations. A promising avenue for insights lies in the coupling of bacterial scRNA-seq with transcriptional regulation analysis, as implemented in our study. The iModulon-based assessments enabled cross-population relationships to be quantified and visualized. Furthermore, trajectory analysis provided another means to understand signaling dynamics, especially when linked to gene expression. While only a subset of genes correlating with the trajectory were discussed, many more remain unexplored (Supplementary Data 8 – 9 ). Several of these genes encode uncharacterized proteins that could potentially play key roles in biofilm formation and may represent attractive anti-biofilm therapeutic or prophylactic targets. An important future direction towards a better understanding of biofilm dynamics is to perform BaSSSh-seq during different stages of biofilm growth to assess 1) temporal alterations in gene expression; 2) changes in transcriptional regulatory networks through iModulons; and 3) clustering patterns during maturation. We did not detect many genes previously identified to be important during biofilm formation, such as the icaABCD and cidAB operons, which is likely because established biofilms were examined in this study 57 , 60 . Relating transcriptionally defined clusters to spatially defined microstructures and regions throughout the various stages of biofilm development would augment our understanding of biofilm growth and signaling, which could be achieved by constructing fluorescent reporters for genes that are enriched in distinct clusters. BaSSSh-seq successfully generated powerful visualizations of biofilm transcriptional regulation paired with gene-level analyses for subpopulation characterization. The heterogeneity and coordinated patterns of gene regulation observed across biofilm clusters overwhelmingly illustrate how the ensemble-averaged expression from traditional bulk RNA-seq is insufficient. Accordingly, single-cell resolution also provides quantitative information on relative population sizes, a metric that is lost in bulk methods. Although many biofilm cells displayed a transcriptionally dormant phenotype (cluster 0), we focused our efforts on more active biofilm populations and how they interacted with the immune response. Our analyses demonstrated that biofilm undergoes dramatic transcriptional alterations that are tailored to the immune cell encountered. Although speculative, it is intriguing to consider that the most metabolically active biofilm clusters were responsive to MΦ and PMN challenge since these immune populations are major producers of ROS, RNS, and proteases that place strong pressures on bacteria 123 , 148 , 152 , 153 . In contrast, G-MDSCs do not exhibit antibacterial activity, so the biofilm does not need to expend substantial energetic resources to transcriptionally respond to this non-threat 34 , 35 . These findings have significant potential to inform more effective immunomodulatory therapies and support the concept of nutritional immunity described in the literature 168 . Future efforts will move in vivo, to explore the diversity of S. aureus adaptation and immune responses across different tissue niches. Although highly functional, areas for improvement remain throughout the BaSSSh-seq methodology and analyses. For example, the number of barcoded cells with appreciable numbers of mRNA reads in biofilm samples was low. Insights from our comparisons of biofilm and planktonic cultures suggest this results from decreased transcriptional activity within biofilm. Nonetheless, membrane permeabilization conditions prior to barcoding could be more thoroughly studied to improve time and temperature for maximal barcode diffusion and RNA capture. Additionally, the barcoding could be expanded to 384-well plates to increase cell capacity by >60×. Sequencing depth also impacts the capture and detection of low-level transcripts, and with incorporation of rRNA depletion we improved cost efficiency and information content for sequencing runs, permitting usage of a mid-output kit on an Illumina NextSeq 500/550 series platform. However, availability of larger sequencers and kits exist for increasing sequencing depth >200×. As discussed further in the Methods, an inherent background noise exists, evident in the UMAP overlays in Figs. 2 – 5 where many genes were expressed at baseline levels throughout all clusters. This limitation restricted the statistical power of some analyses, and improvements would allow for higher confidence in identifying targets for experimental validation. Reduction in noise levels could be realized through adjustment of randomer concentrations in both reverse transcription and second strand synthesis steps, fragmentation conditions used in library prep, and/or modification of alignment parameters. Clustering itself could be further optimized to identify more meaningful classifications through further adjustments to parameter settings and/or future advances in clustering tools and algorithms. From a technical perspective, exploration of long-read sequencing presents a promising avenue that would allow fragmentation to be bypassed, leading to substantial noise reduction while potentially providing new insights into large-scale operon architecture. Several limitations are also evident from an experimental standpoint. First, as noted above, this study examined mature biofilm to assess how various immune cell subsets altered transcriptional programs. Performing BaSSSh-seq at regular intervals during biofilm development could provide new insights into fundamental populations that expand at key steps (i.e., attachment, exodus, and expansion) 53 . Second, spatial information about how specific biofilm transcriptional clusters relate to structural attributes (i.e., attachment, tower formation) is an interesting area to pursue as the resolution of spatial transcriptomic approaches improve. Based on the nature of this work describing BaSSSh-seq as a resource, the importance of specific S. aureus genes in biofilm biology or metabolism were not assessed, although we did validate changes in biofilm metabolism, respiration, and ROS as an initial step. Additionally, only one co-culture interval of biofilm and immune cells was examined (2 h) as a proof-of-concept for biofilm adaptation; however, the kinetics of these changes could be explored in future studies. Finally, in vitro biofilms grown in RPMI-based culture medium on coated plate surfaces do not replicate complex infection environments in vivo. While RPMI-based medium was necessary for leukocyte compatibility 169 – 171 , differences in glucose levels and other nutrients, as well as surface properties, are unable to model the full diversity of conditions encountered within the host. This further motivates the need to expand applications in vivo where differences in biofilm transcriptional profiles are expected in a niche-dependent manner based on nutrient availability and surface composition 172 . Overall, the BaSSSh-seq method coupled with powerful computational approaches facilitates the high-throughput study of biofilm transcriptional heterogeneity at a new resolution. The datasets provide a rich resource for the biofilm community to explore, and the optimized protocols and analyses provide a mechanism to aid in identification of new therapeutic targets and strategies."
} | 4,523 |
36426747 | null | s2 | 4,161 | {
"abstract": "Depletion attractions, occurring between surfaces immersed in a polymer solution, drive bacteria adhesion to a variety of surfaces. The latter include the surfaces of non-fouling coatings such as hydrated polyethylene glycol (PEG) layers but also, as demonstrated in this work, surfaces that are bacteria-adhesive, such as that of glass. Employing a flagella free "
} | 91 |
30699163 | PMC6353167 | pmc | 4,163 | {
"abstract": "Over the past several decades, coral reef ecosystems have experienced recurring bleaching events. These events were predominantly caused by thermal anomalies, which vary widely in terms of severity and spatio-temporal distribution. Acropora corals, highly prominent contributors to the structural complexity of Pacific coral reefs, are sensitive to thermal stress. Response of Acropora corals to extremely high temperature has been well documented. However, studies on the effects of moderately high temperature on Acropora corals are limited. In the summer of 2016, a moderate coral bleaching event due to moderately high temperature was observed around Sesoko Island, Okinawa, Japan. The objective of this study was to examine thermal tolerance patterns of Acropora corals, across reefs with low to moderate thermal exposure (degree heating weeks ~2–5°C week). Field surveys on permanent plots were conducted from October 2015 to April 2017 to compare the population dynamics of adult Acropora corals 6 months before and after the bleaching events around Sesoko Island. Variability in thermal stress response was driven primarily by the degree of thermal stress. Wave action and turbidity may have mediated the thermal stress. Tabular and digitate coral morphologies were the most tolerant and susceptible to thermal stress, respectively. Growth inhibition after bleaching was more pronounced in the larger digitate and corymbose coral morphologies. This study indicates that Acropora populations around Sesoko Island can tolerate short-term, moderate thermal challenges.",
"conclusion": "Conclusion Over the past decades, trait-based approaches, i.e., studying traits of corals such as growth forms, colony size, and growth rate, among coral genera or higher taxa have gained recognition in coral reef ecology [ 81 ]. A recent metanalysis showed coral morphology to be a reliable predictor of bleaching variability [ 82 ]. However, studies examining intrageneric variability are limited, and the thermal response within the genus Acropora is usually inconsistent across studies. For example, digitate Acropora were the most thermally sensitive in some studies [ 26 , 83 ], but tabular Acropora were found to be more sensitive in other studies [ 14 , 84 , 85 ]. Such inconstancy might suggest that the local environment, traits of the species studied, or some other factors had roles in governing intrageneric variability. Therefore, it is important to conduct studies of coral bleaching across different environments and temperatures to delineate the roles of morphology, environment, and species-level traits in intrageneric thermal response variability. In conclusion, our first hypothesis, that bleaching prevalence is driven primarily by thermal exposure, was supported in the present study. Our second hypothesis, that demographic rates recover to normal levels after bleaching was, however, not. Our third hypothesis, that morphological traits of colonies explain differences in thermal exposure response, was also accepted as indicated by the size specific thermal response and morphological thermal hierarchy observed in this study. Overall, future studies investigating the relationships between multiple morphological traits, quantified environmental conditions, and demographic rates can be informative regarding how coral reefs of Sesoko Island, Japan will respond to future climate change.",
"introduction": "Introduction Coral reef ecosystems worldwide are being challenged by increasing global and local anthropogenic stress. Stressors can affect individual performance, community species composition, and consequently, ecosystem function [ 1 – 4 ]. Coral bleaching is caused by the collapse of the mutualistic relationship between host corals and their symbiotic algae and it is a major threat to the health and survival of coral reefs. Bleaching occurs mainly in response to rising average sea surface temperature (SST) with strong irradiance [ 5 – 8 ]. Bleached corals are physiologically stressed and this affects growth and mortality in coral populations [ 9 , 10 ]. Consequently, coral bleaching may transform the structure and functional diversity of coral communities [ 11 ]. In addition to its immediate effects, bleaching may also have long-term effects on corals after temperatures have returned to normal. Omori et al. [ 12 ] reported that the fertilization rates of Acropora corals decreased by ~50% in 1999 following the 1998 mass bleaching event on Aka Island in the Ryukyus. The bleaching event may have reduced sperm motility. Ward et al. [ 13 ] also reported zero reproductive output of both bleached and recovered corals on Heron Island, Great Barrier Reef (GBR), after the 1998 bleaching event. Muko et al. [ 14 ] found that the coral recruitment rates after bleaching events were lower than those before on Iriomote Island in the Ryukyus. The prolonged effects of bleaching on coral growth are inconsistent. Some studies reported reduced coral growth after a thermal anomaly [ 15 ], while others indicated that the growth rates of surviving colonies were unaffected by bleaching [ 14 ]. High coral mortality rates are typical immediately following a severe bleaching event [ 10 , 16 – 18 ]. In contrast, extended or prolonged coral mortality 6–8 months after a bleaching event was observed at the GBR [ 11 ]. Understanding the risks and mechanisms of the long-term prolonged effects of bleaching on coral populations will help us to predict future shifts in coral community health and functioning. Many coral reefs globally, including those in the Ryukyu Islands of Japan, experienced a severe bleaching event in 1998 [ 19 ]. At Sesoko Island, the corals were severely affected by this bleaching event [ 20 ]; up to 85% of the hard and soft coral cover was lost. The massive hard coral morphologies, like Porites , were the survivors, whereas the branching hard coral morphologies such as Acropora and the pocilloporids were more severely affected [ 20 ]. Similar morphology-specific bleaching susceptibility has been reported for other coral reefs [ 21 , 22 ]. In 2016, severe bleaching events (>60% corals bleached) occurred on many reefs worldwide [ 11 ], including the Ryukyu Islands [ 23 ]. Nevertheless, bleaching-induced mortality of Acropora corals (thermally vulnerable taxa in the 1998 bleaching event) [ 20 ] was lower in 2016 than it was in 1998 on the Sesoko Island reef; all Acropora colonies larger than 10 cm in diameter died during the 1998 bleaching event [ 20 ]. In this study, we observed the effects of moderate thermal stress on branching Acropora corals. This genus dominates in many reefs in the Ryukyu Islands. Its member species show high morphological diversity and provide a three-dimensional habitat for other reef organisms. The life history and morphological traits of corals may determine their thermal stress tolerance. It has been postulated that compared to fast-growing branching species, slow-growing massive species have higher thermal tolerance [ 11 , 22 , 24 – 26 ]. Morphological traits have been assessed at the polyp and colony levels. The thicker polyp tissue of massive corals compared to that of branching corals provides shade for the symbiotic algae within the coral cells via polyp tissue retraction. This feature may, in part, account for the relatively higher thermal tolerance of massive corals [ 19 , 20 , 27 ]. At the colony level, interspecific variations like encrusting vs. branched colonies and intraspecific variation such as small vs. large colonies have been discussed in terms of their relative differences in mass flux rate. High mass flux rates are associated with the efficient removal of oxidative metabolites by diffusion [ 20 , 28 ]. Acropora corals most commonly have a branching colony morphology. However the genus is morphologically diverse and includes corymbose, digitate, tabular, and arborescent forms [ 29 ]. In this study, we excluded tissue thickness from the discussion of thermal tolerance differences because all Acropora corals have similar tissue thickness [ 20 ]. We therefore evaluated the effects of colony morphology and growth on thermal stress tolerance among various Acropora species. We also compared the effects of thermal stress after bleaching events on the growth of colonies of different sizes within the same species. Seawater temperature, cloud cover, wind force, seawater turbidity, reef microhabitat structure (such as coral overhang and crevices), water flow, wave action, and depth may all reduce thermal stress and cause coral bleaching response heterogeneity on a small spatial scale (≤tens of km) [ 21 , 30 – 33 ]. Degree heating weeks (DHW) is an index of accumulated heat exposure over 12 weeks [ 34 – 37 ], and therefore it considers both intensity and duration of thermal exposure. DHW of 4°C-week usually results in significant bleaching and 8°C-week results in critical, wide-spread bleaching and significant mortality [ 35 ]. Following these criteria, DHW between 4–8°C-week are defined as representing a moderate thermal anomaly in this study. DHW has been widely used to quantify bleaching thresholds and to asses thermal stress variability on a large spatial scale (≥hundreds of km) [ 38 – 40 ]. Small-scale thermal disparity and consequent differential bleaching responses have been observed [ 33 , 41 ]. Nevertheless, to the best of our knowledge, no studies have used DHW to determine thermal exposure variability on a small spatial scale. In the present study, we explored the prolonged or extended effects of bleaching in Acropora corals. We compared Acropora coral population dynamics before and after a moderate thermal anomaly in different environmental regimes and examined whether (1) bleaching prevalence is driven primarily by the degree of thermal exposure, (2) Acropora demographic rates recover after the temperature returns to normal, and (3) Acropora colony morphological traits determine inter and intraspecific differences in thermal stress tolerance.",
"discussion": "Discussion The results of the present study indicate that the degree of coral bleaching may vary among reefs within a small spatial range, such as several kilometers, primarily owing to the relative differences in thermal exposure among reefs (Figs 2 , 3 and S2 ). The maximum daily temperature and daily temperature fluctuations were significantly higher at Sesoko Station than at all other sites within a 5 km range ( S2 Fig and S3 File ), in addition the DHW at Sesoko Station was also highest ( Fig 2 ). Compared to other shallow sites (Hamamoto and Yakkai) Sesoko Station was also closest to the shore ( Table 1 ). This might explain the higher temperature regime at this site. Sesoko Station was the only site where all Acropora colonies were bleached irrespective of their morphology in the summer of 2016 ( Fig 3 ). Bleaching-induced mortality and suppressed growth rates were observed for all morphologies following the bleaching event only at Sesoko Station. Relative to global bleaching events, Sesoko station was exposed to moderate thermal stress [ 4 , 23 ]. Moderate thermal anomalies are known to elicit a stress response in corals. For example, some sites at Florida Keys experiencing DHW ≤3°C-weeks resulted in a loss of 5% Shannon diversity [ 57 ]. Moderate temperature anomalies (+ 1.8°C) at Aka Island, Okinawa, Japan resulted in narrower size-class distribution of corymbose Acropora due to size specific mortality rates [ 58 ]. Our study additionally showed that, in the absence of a local environment filter, even mild to moderate thermal anomalies can result in prolonged effects such as depressed growth rates and increased mortality rates. Variations on a small spatial scale may have implications for the local conservation of coral reefs in the Anthropocene (sensu [ 4 ]). To preserve coral larval sources and sinks, corals located on reefs where the temperature is low, which are remote from local disturbances like crown of thorns starfish (COTS) predation and construction activity should be selected for the conservation. Prolonged mortality was observed in the digitate colonies at Sesoko Station after the 2016 bleaching event even when temperature had already returned to normal. The mortality rates immediately after bleaching were both size- and morphology-independent at Sesoko Station. Six months after bleaching, mortality of the digitate colonies continued at this site. Prolonged mortality in the digitate colonies may have been the result of negative growth or partial mortality, which occurred in all the bleached digitate colonies there. The physiological processes impaired by bleaching caused whole or partial coral colony mortality [ 6 , 59 – 61 ]. Susceptibility to bioerosion may increase in partially dead colonies [ 62 ]. Colonization of turf algae on the dead skeletons in partially dead coral colonies may increase microbial activity and degrade the local environment [ 63 ]. The mass flux rates determined by colony morphology might explain the different responses to thermal exposure among the various types of Acropora corals in this study. Digitate and corymbose colonies had decreased growth rates in t2 at both Sesoko Station and West Sesoko, while that of tabular colonies decreased only at Sesoko Station. Furthermore, extended mortality was observed only for digitate colonies, indicating that digitate and tabular colonies were the most susceptible and resistant to thermal stress, respectively. Some studies have suggested that fast-growing corals with high metabolic rates are relatively more sensitive to thermal anomalies because they accumulate harmful bleaching by-products such as reactive oxygen species (ROS) [ 10 , 11 , 24 – 26 , 64 ]. Growth was slower in digitate colonies than in corymbose and tabular colonies; however, bleaching susceptibility was the greatest in digitate colonies. In contrast to the fast growth hypothesis, mass transfer coefficients of various geometric shapes based on Reynolds-Sherwood numbers calculated by Patterson [ 65 ], may corroborate the order of thermal sensitivity in aquatic invertebrates. The relative differences in thermal susceptibility among the colony morphologies observed in the present study followed Patterson’s mass transfer theory ( Table 7 ), with the highest mass flux rates for flat shapes like tabular morphology. Furthermore, digitate colonies had the lowest VRs although they did not significantly differ from those for tabular colonies. The presence of such a pattern, albeit weak, suggests that morphological traits of corals may be associated with enhanced mass flux in branched corals [ 45 ]. Size-specific growth decline in response to size-specific mass flux rates may decrease mean corymbose and digitate colony size due to climate change. After the bleaching event investigated in the present study, the growth of larger corymbose and digitate colonies decreased more than it did for the smaller ones. Partial mortality is more likely to occur in larger colonies at both normal [ 66 , 67 ] and high [ 58 , 68 , 69 ] temperatures. Several studies have shown that smaller colonies were comparatively less affected by high temperature exposure than larger colonies of the same species [ 20 , 70 , 71 ]. These observations were ascribed principally to the more effective removal of harmful metabolites like ROS because of the relatively higher mass flux in small, flat coral colonies [ 20 , 28 ]. Edmunds and Burges [ 72 ] empirically determined that high temperature has more severe negative effects on photosynthesis and respiration in larger whole branching Pocillopora verrucosa than it does on smaller ones. These physiological responses, therefore, could also influence coral growth. The size-nonspecific responses of tabular colonies to thermal anomalies observed in the present study may be explained by the fact that tabular colony branch height increases only slightly as the colonies grow. In contrast, height significantly increases as corymbose and digitate colonies grow. Coral colonies with large height to diameter (aspect) ratios have comparatively lower mass flux rates [ 28 ], therefore, increasing the aspect ratio with the growth of corymbose and digitate morphologies might explain the observed size-specific thermal responses. Corals at South Sesoko may have escaped thermal stress or quickly recovered using efficient mass transfer. The growth of larger corymbose and digitate colonies also decreased at South Sesoko. Nevertheless, this site was not as severely affected by thermal anomalies as the other sites. Moreover, this site experienced high partial mortality (Figs 4 and 6 ) and mortality rates even in t1, suggesting that factors other than temperature were involved here. At all sites, number tags were attached by cable ties to iron rods and used to mark fixed plots. These loosened only at South Sesoko, suggesting that the water movement was strongest at this site. Field studies after the 1998 mass bleaching event reported relatively milder bleaching effect at sites with high water flow [ 20 , 73 ]. High water flow may mitigate coral bleaching by lowering oxidative stress through efficient mass flux [ 30 , 74 ]. Experimental studies also validated that mass transfer in branched corals was higher under oscillatory flow (wave action) than it was under unidirectional flow [ 45 ]. This hypothesis should be tested by quantifying water movement in future studies. High turbidity and increased heterotrophy during and after thermal exposure may have contributed to thermal resistance at Hamamoto and Yakkai. Corals were not thermally stressed at this turbid site. High turbidity alleviates the thermal and solar irradiance effect [ 75 – 77 ] possibly by reducing solar irradiance. In addition, heterotrophic plasticity in some species might acclimatize them to turbid environments by increasing their feeding rate [ 78 ]. Increased feeding rate in thermally stressed corals [ 79 ] coupled with high organic and nutrient load in turbid environments would lead to higher lipid content enabling corals to maintain their growth and survival rates following a bleaching event [ 80 ]. It is possible that Acropora at Hamamoto and Yakkai were not thermally stressed due to a combination of the above factors."
} | 4,580 |
26346879 | PMC4560930 | pmc | 4,164 | {
"abstract": "Background Purified terephthalic acid (PTA) wastewater from a petrochemical complex was utilized as a fuel in the anode of a microbial fuel cell (MFC). Effects of two important parameters including different dilutions of the PTA wastewater and pH on the performance of the MFC were investigated. Methods The MFC used was a membrane-less single chamber consisted of a stainless steel mesh as anode electrode and a carbon cloth as cathode electrode. Both power density and current density were calculated based on the projected surface area of the cathode electrode. Power density curve method was used to specify maximum power density and internal resistance of the MFC. Results Using 10-times, 4-times and 2-times diluted wastewater as well as the raw wastewater resulted in the maximum power density of 10.5, 43.3, 55.5 and 65.6 mW m −2 , respectively. The difference between the power densities at two successive concentrations of the wastewater was considerable in the ohmic resistance zone. It was also observed that voltage vs. initial wastewater concentration follows a Monod-type equation at a specific external resistance in the ohmic zone. MFC performance at three different pH values (5.5, 7.0 and 8.5) was evaluated. The power generated at pH 8.5 was higher for 40% and 66% than that for pH 7.0 and pH 5.4, respectively. Conclusions The best performance of the examined MFC for industrial applications is achievable using the raw wastewater and under alkaline or neutralized condition.",
"conclusion": "Conclusion The main purpose of this research was to provide more information and insight into the MFC operation that can pave the way towards practical utilization of MFC technology for the application of real wastewater. Bioelectricity generation using purified terephthalic acid wastewater from a petrochemical plant was successfully conducted in a single chamber microbial fuel cell with a stainless steel mesh as anode electrode. The influence of wastewater concentration on the MFC performance showed that using the raw wastewater with the concentration of 8000 mg COD L −1 results in the highest power density (65.6 mW m −2 ). Our observations suggest that the best performance is achievable when the MFC operates at the ohmic zone and has an external resistance which is equal to the internal resistance of the cell. The voltage against different initial concentrations of the wastewater in the ohmic zone followed a Monod-type equation. This observation implies that the concentration increase has a positive effect of electricity generation but it cannot exceed a maximum value. Performance of the MFC at different pH values was investigated and the highest power density was observed under alkaline condition (pH 8.5) due to inactivation of acidogenic and methanogenic bacteria in favor of more activity for electrogenic bacteria.",
"introduction": "Introduction Microbial fuel cell (MFC) is a device that converts biochemically released energy from bacterial catalysis of organic and inorganic materials into electrical energy. In MFCs, electricity generation and wastewater treatment can occur, simultaneously. Therefore, MFCs are considered as one of the potential solutions to overcome the crises of energy shortage and environmental pollution. On the other hand, many challenges are still remained for commercialization of MFC technology. Designing a cost-effective system with high power generation is one of the most important challenges. For example, in order to achieve a high performance, expensive materials such as platinum as cathodic catalyst, carbon cloth as cathode or anode electrode, and also a suitable membrane such as nafion are inevitably necessary. Accordingly, more studies are required to find efficient materials both in terms of cost and power production [ 1 ]. In recent years, this technology has been studied by many researchers. Generally, different parameters including both operational and designing factors affect the MFC performance. Nature of the substrate, substrate concentration [ 2 ], temperature [ 3 ], microorganisms’ species, alkalinity of anode and cathode chambers [ 4 ], external resistance [ 5 ] and residence time [ 6 ] are among the operational parameters. Anode and cathode material [ 7 ], type of membrane [ 8 ] and MFC architecture are among the designing parameters. A wide range of different wastewaters have been examined in MFCs. For instance, domestic wastewater [ 3 , 9 ], landfill leachate [ 6 ], coking wastewater [ 2 ], confectionery wastewater [ 8 ] and cassava mill wastewater [ 10 ] can be mentioned. However, wastewaters from petrochemical industries have not been receiving much attention due to complications in their biodegradatiuon. Purified terephthalic acid (PTA) wastewater with a high strength organic content is generated during the production process. PTA is a raw material for manufacturing of many petrochemical products such as polyester textile fibers, polyethylene terephthalate bottles and polyester films. As much as 3–10 m 3 of such a wastewater is usually generated per one ton of PTA as product [ 11 ]. In our previous work, we studied the feasibility of utilizing PTA wastewater in a MFC for the first time [ 12 ]. In the present work, we investigate the effect of two main characteristics of the wastewater i.e. organic load and pH value that significantly influence the power generation. In the present research, we investigated the following issues: Effect of wastewater concentration on power generation Correlation between voltage and wastewater concentration Effect of different pH values on power generation",
"discussion": "Results and discussion Power production at different concentrations of wastewater Effect of substrates’ concentration required for the microbial activity in the anode chamber was investigated. The substrate as fuel was supplied in the following order, consecutively: 10-times diluted wastewater (C 1 ), 4-times diluted wastewater (C 2 ), 2-times diluted wastewater (C 3 ) and the raw wastewater (C 4 ). Figure 2 shows power density curves of the MFC at different concentrations of the wastewater. Maximum power density was 10.5, 43.3, 55.5 and 65.6 mW m −2 for C 1 , C 2 , C 3 and C 4 , respectively. Internal resistance of the MFC was 5.6 kΩ for all concentrations except for C 3 that was equal to 3.2 kΩ. Figure 2 \n Power density curves at different concentration of PTA wastewater. Power density curves were obtained when the voltage reached to a stable value. Maximum power densities of C 3 , C 2 and C 1 wastewaters were 84%, 66% and 16% of the maximum power density of the raw wastewater. Therefore, the maximum power density increased when more concentrated wastewater was used. Current generated in a MFC is limited by two factors: (i) oxidization rate of substrate by bacteria and (ii) rate of electrons transfer to the electrode surface [ 1 ]. Substrate oxidation rate depends on the concentration of substrate which is assumed usually as a first order reaction. Therefore, it is expected to observe a higher current and power at higher concentrations. However, other factors such as mass transfer and biofilm layer thickness can suppress power production. The influence of such factors can be studied when the substrate concentration is high enough where oxidation rate is not limited. This observation has been further explained in the next section. Power differences at different external resistances between two successive concentrations Increase in the power density due to employing more concentrated wastewater in the anode, was correlated to the external resistance. Figure 3 indicates the difference between the power densities at two successive concentrations of wastewater at definite external resistances. The maximum power density difference was 32.7, 11.9 and 18.3 mW m −2 between C 2 - C 1 , C 3 - C 2, and C 4 - C 3 , respectively. Figure 3 \n Power density difference between two successive concentrations of wastewater at different external resistances. Maximum power density was 10.5, 43.3, 55.5 and 65.6 mW m −2 for C 1 , C 2 , C 3 and C 4 , respectively. The wastewater had the concentration of 8000 mg COD L −1 . According to the polarization curve, mass transfer resistance zone and activation loss zone are observable at low external resistances and large external resistances, respectively. All three curves follow the same trend. The power density difference was negligible at higher external resistances while the cell operated in the activation loss zone. This is the same at very low external resistances when the cell operates in the mass transfer zone. This shows that the substrate concentration has a minor effect in these zones. Accordingly, if a MFC is working with an external resistance which leads to operating, either in the activation loss zone or the mass transfer zone, increasing the substrate concentration would not have a significant effect on its performance. In contrary, a considerable power density difference was observed in the ohmic resistance zone. The power density difference reached a maximum value at an external resistance equal or close to the internal resistance of the MFC. Therefore, according to these observations, the maximum power production is reachable when the external resistance is near the internal resistance. Besides, the major positive effect of concentration increase is visible when the MFC works in the ohmic zone. Generated voltage versus wastewater concentration in ohmic zone In this section, the behavior of MFC in the ohmic zone is explored. The generated voltage at different concentration of wastewater for a certain external resistance (in the Ohmic zone) is exhibited in Figure 4 . It was observed that the correlation between voltage and concentration is so that at higher concentrations, a little increase occurs in the voltage generation. For example, there is no significant difference between the produced, voltage when C3 or C4 was applied. One can say that the concentration increase effect is limited even in the ohmic zone which might be as a result of concentration inhibition that halts bacteria metabolism. Figure 4 \n Generated voltage at different concentration of wastewater. Monod-type behavior of voltage against concentration. The voltages vs. concentration curves suggest a Monod-type equation as follows: 1 \\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}$$ V={V}_{\\max}\\frac{s}{K_s+s} $$\\end{document} V = V max s K s + s where, V (mV) and V max (mV) stand for voltage and maximum voltage, respectively; S (mg L -1 ) represents COD of substrates and K s (mg L −1 ) is the half-saturation constant. V max and K s were calculated for each curve as presented in Table 1 . Table 1 \n Calculated constants of Eq. \n 1 \n for different external resistances \n \n R \n ext \n (kΩ) \n \n V \n max \n (mV) \n \n K \n s \n (mg L \n -1 \n ) \n 1 45.0 1148.4 3 98.0 48.0 5 130.5 614.7 Maximum achievable voltage versus external resistance follows a linear equation: 2 \\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}$$ {V}_{\\max }=21.375\\ {\\mathrm{R}}_{ext}+27 $$\\end{document} V max = 21.375 R e x t + 27 Not to be neglected that the above equation is valid only when the MFC operates at the ohmic zone. Power production at different pH values pH has a significant effect on the activity of bacteria in terms of removal efficiency and energy production. In order to study the influence of pH, the MFC was fed with 10- times diluted wastewater at three different pH values including 8.5, 7.0 and 5.4, periodically. These pH values were selected based on the optimal range of the pH reported for methane-producing bacteria. It has been observed that these bacteria are active in the pH range of 6.3-7.8 [ 14 ]. Presence of methane producers is very possible in our system. The power density curves for different pH values are shown in Figure 5 . It was observed that the maximum power density was 12.5, 7.5 and 4.3 mW m −2 for the pH values of 8.5, 7.0 and 5.4, respectively. Figure 5 \n Power density curves at different pH values for 10-times diluted wastewater. \n In general, the higher the pH value, the higher the power density. The produced power at pH 8.5 was higher for 40% and 66% than that for pH 7.0 and pH 5.4, respectively. This observation is consistent with other previous studies [ 15 , 16 ]. Apparently, acidogenic bacteria are active in pH 5.5. Under this condition, hydrogen production would be the dominant mechanism which overcomes the pollutants degradation and a decreased removal rate is expected compared to the neutral or alkaline conditions [ 14 ]. Due to the low removal rate, fewer electrons are released and the power production is lowered, consequently. At pH 7.0, methane gas production is the dominant metabolic pathway. This would lead to a less number of released electrons that can contribute in electricity generation and a lower power density is observed, eventually. The increase in power density production at pH 8.5 might be due to the lower activity of methanogenic and acidogenic bacteria. As a result, the electrons released in the oxidation process of the substrates would contribute significantly in electricity generation. However, further studies are required to clarify the occurrence of these phenomena, more precisely. It can be concluded from the trend of power production at different pH values that alkaline condition provides a favorable situation for the growth of electrogenic bacteria. Previous studies have shown that the electrochemical interaction of bacteria significantly increases under alkaline conditions [ 15 , 16 ], which ultimately leads to a higher power production."
} | 3,520 |
37439151 | PMC10502755 | pmc | 4,166 | {
"abstract": "Summary The photosynthetic light reaction in cyanobacteria constitutes a highly attractive tool for productive biocatalysis, as it can provide redox reactions with high‐energy reduction equivalents using sunlight and water as sources of energy and electrons, respectively. Here, we describe the first artificial light‐driven redox cascade in Synechocystis sp. PCC 6803 to convert cyclohexanone to the polymer building block 6‐hydroxyhexanoic acid (6‐HA). Co‐expression of a Baeyer‐Villiger monooxygenase (BVMO) and a lactonase, both from Acidovorax sp. CHX100, enabled this two‐step conversion with an activity of up to 63.1 ± 1.0 U/g CDW without accumulating inhibitory ε‐caprolactone. Thereby, one of the key limitations of biocatalytic reactions, that is, reactant inhibition or toxicity, was overcome. In 2 L stirred‐tank‐photobioreactors, the process could be stabilized for 48 h, forming 23.50 ± 0.84 m m (3.11 ± 0.12 g/L) 6‐HA. The high specificity enabling a product yield ( Y \n P/S ) of 0.96 ± 0.01 mol/mol and the remarkable biocatalyst‐related yield of 3.71 ± 0.21 g 6‐HA /g CDW illustrate the potential of producing this non‐toxic product in a synthetic cascade. The fine‐tuning of the energy burden on the catalyst was found to be crucial, which indicates a limitation by the metabolic capacity of the cells possibly being compromised by biocatalysis‐related reductant withdrawal. Intriguingly, energy balancing revealed that the biotransformation could tap surplus electrons derived from the photosynthetic light reaction and thereby relieve photosynthetic sink limitation. This study shows the feasibility of light‐driven biocatalytic cascade operation in cyanobacteria and highlights respective metabolic limitations and engineering targets to unleash the full potential of photosynthesis.",
"introduction": "Introduction Photosynthetic microorganisms have gained increasing attention as host organisms for biotechnology (Jodlbauer et al ., 2021 ). Relying on water, CO 2 and light as the main resources, their phototrophic metabolism has a high potential to enable truly sustainable production processes. Especially cyanobacteria have been studied and discussed as workhorses for the production of (bulk) chemicals such as ethanol (Gao et al ., 2012 ), 1‐butanol (Liu et al ., 2019 ), hydrogen (H 2 ) (Appel et al ., 2020 ), glycerol (Savakis et al ., 2015 ), to name only a few. Since recently, phototrophic microorganisms are studied for redox biotransformations, thereby making more efficient use of photosynthesis as compared to biomass formation via the direct coupling of biocatalytic electron/energy sinks to energy source utilization, that is, the photosynthetic light reaction (Barber, 2009 ). Redox biotransformations are especially attractive since the separately added biotransformation substrate does not directly interfere with the host C‐metabolism (Theodosiou et al ., 2022 ; Toepel et al ., 2023 ). Thereby, these biotransformations function as (external) sinks utilizing photosynthesis‐derived energy—in the form of reduction equivalents—that may not be used by the CBB cycle due to its lower turnover capacity compared to the light reaction. Indeed, sink engineering has been shown to relieve sink limitation of the photosynthetic metabolism tapping the unused potential of photosynthesis (Berepiki et al ., 2016 ; Grund et al ., 2019 ). Oxygenases are an especially interesting enzyme class for photo‐biocatalysis, as the photosynthetic light reaction does not only supply reduction equivalents but also O 2 \n in situ (Hoschek et al ., 2017 ), overcoming typical limitations experienced with heterotrophs, that is, the addition and efficient utilization of reduction equivalent‐delivering co‐substrates or O 2 mass transfer (Kadisch et al ., 2017 ). Cytochrome P450 monooxygenases (CYPs) (Berepiki et al ., 2018 ; Hoschek et al ., 2019 ; Lassen et al ., 2014 ; Wlodarczyk et al ., 2015 ) and Baeyer‐Villiger monooxygenases (BVMOs) (Erdem et al ., 2022 ; Tüllinghoff et al ., 2022 ) have been applied in the model strain Synechocystis sp. PCC 6803 ( Synechocystis ), yielding specific activities up to 39.2 ± 0.7 (Hoschek et al ., 2019 ) and 60.9 ± 1.0 U/g CDW (Tüllinghoff et al ., 2022 ) (1 U = 1 μmol/min) corresponding to volumetric productivities of 2.35 ± 0.04 and 3.40 ± 0.06 m m /h, respectively. Several studies revealed that the main limitations of light‐driven whole‐cell redox biocatalysis are: (i) light availability, (ii) metabolic constraints and (iii) reactant toxicity and inhibition (Erdem et al ., 2022 ; Hoschek et al ., 2019 ; Tüllinghoff et al ., 2022 ). With light being the energy source to drive heterologous redox reactions as well as the host metabolism, it is obvious that light limitation has to be avoided (Hobisch et al ., 2021 ). In a recent study involving a BVMO from Burkholderia xenovorans , insufficient light supply was concluded to limit the (light‐driven) conversion of cyclohexanone (C‐one) to ε‐caprolactone (ε‐cl) (Erdem et al ., 2022 ). In our previous study employing a BVMO from Acidovorax sp. CHX100 ( Acidovorax ), however, 2.4‐fold higher activities were achieved at a larger scale (2 L stirred‐tank‐photobioreactor vs. mL‐scale), with product‐related effects and not light as the decisive limitation (Tüllinghoff et al ., 2022 ). Thereby, the product ε‐cl did not inhibit the enzyme, but led to cell toxification and thereby inhibited the whole‐cell biocatalyst. Such reactant inhibition and toxicity are well known to impair biocatalyst viability and stability, be it on the metabolic, physiological, or enzymatic level (Fürst et al ., 2019 ; Scherkus et al ., 2017 ; Schmidt and Bornscheuer, 2020 ). With the highly active reductase Yqjm in Synechocystis , initial specific activities of up to 170 U/g CDW have been reported, but the toxicity of the product 2‐methylsuccinimide is arguably thwarting this reaction (Assil‐Companioni et al ., 2020 ). As an important metabolic constraint, the ATP:NADPH ratio may be disturbed upon electron withdrawal for redox biocatalysis. Lately, it has been shown that careful balancing of this ratio by simultaneously applying an ATP sink can benefit redox biocatalysis (Santos‐Merino et al ., 2021 ). In this study, we constructed an enzyme cascade in Synechocystis for the conversion of C‐one to non‐toxic 6‐hydroxyhexanoic acid (6‐HA), an equally interesting synthon and polymer building block, thereby minimizing reactant effects on host metabolism. Recently, Acidovorax BVMO has been used in Pseudomonas taiwanensis VLB120 as part of cascades converting cyclohexane to 6‐HA (Schäfer et al ., 2020 ), 6‐amino‐hexanoic acid (Bretschneider et al ., 2021 ), and adipic acid (Bretschneider et al ., 2022 ), involving an Acidovorax lactonase to convert ε‐cl to 6‐HA, which also was used in this study. So far, enzyme cascades applied in cyanobacteria typically involved native carbon fixation (Angermayr et al ., 2015 ; Miao et al ., 2020 ). Examples of enzyme cascading in cyanobacteria making use of non‐native substrates and electrons derived from photosynthetic water oxidation are rare. In a proof‐of‐concept study, Wlodarczyk et al . ( 2015 ) implemented the dhurrin pathway from Sorghum bicolor by expressing two membrane‐bound cytochrome P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase. Vector‐based co‐expression of all three genes was demonstrated and the process was translated to 8 L photo‐bioreactors. The final dhurrin titre of 3.34 mg/L was not optimized in the scope of the study. Besides overcoming toxicity‐related limitations, the present study aims at elucidating the capacity of and limitations within cyanobacteria for photosynthesis‐driven in vivo cascades. The approach included the functional co‐expression of BVMO and lactonase genes in Synechocystis and the evaluation and optimization of the resulting cascade regarding inhibition avoidance as well as conversion rate and product titre.",
"discussion": "Discussion By the establishment of efficient photosynthesis‐driven cascade biocatalysis, inter alia to avoid product inhibition and the evaluation of respective inherent metabolic capacity potentials and limitations within cyanobacteria, this study gives crucial insights for future research and developments in the rapidly evolving field of photo‐biotechnology. A two‐step cascade was established in Synechocystis to convert C‐one via ε‐cl to 6‐HA utilizing a BVMO and a lactonase from Acidovorax sp. CHX100. The high activity of the lactonase enabled immediate conversion of ε‐cl upon its formation, thereby preventing product inhibition. These results correspond well to results obtained with these enzymes in heterotrophic P. taiwanensis VLB120 (Schäfer et al ., 2020 ) and are particularly promising, as they illustrate the transferability of engineering principles from heterotrophic to phototrophic chassis strains. The transfer of the photosynthesis‐driven two‐step cascade onto a 2 L scale using photo‐STRs showed that the avoidance of detrimental ε‐cl effects alone did not suffice to stabilize the C‐one conversion over an extended period of time (≥24 h). The exploited specific activity had to be limited by an appropriate substrate feed regime to enable such stabilization. Energy balancing revealed that the moderate feed regime (enabling 10 U/g CDW ) translates to the withdrawal of roughly 16% of intracellular reductant. Withdrawing this share appeared to be feasible from a physiological perspective, as not only the biotransformation rate was stabilized, but also biomass formation occurred to the same (or even slightly higher) extent as without biotransformation. However, a higher share of intracellular reductant could only be withdrawn from cellular metabolism for a few hours, followed by a decrease in both biomass formation and bioconversion rates. Obviously, strong and enduring electron withdrawal affected cell physiology. Thus, the high capacity for light reaction‐derived electron withdrawal observed in the short term appears to be significantly lower in the long term. The question arises, why this is the case and what exactly causes the observed limitation and also the observed decrease in reductant supply by the photosynthetic light reaction. One obvious reason may be that reductant supply by photosynthesis is or becomes limited, either due to external factors like light limitation or restricted metabolic/photosynthetic capacity. Previous studies on Synechocystis expressing reductant‐dependent enzymes elucidated that respective reaction rates are light‐dependent (Hobisch et al ., 2021 ; Hoschek et al ., 2019 ; Tüllinghoff et al ., 2022 ), but also showed that light limitation could largely be relieved at high light intensities (Theodosiou et al ., 2022 ). In the long term, however, increasing the light intensity did not enable a recovery of decreasing C‐one biotransformation activities (Figure 2 ). On the other side, photosynthetic metabolism may suffer from a strong reductant withdrawal by electron‐demanding biotransformations disturbing the NADPH:ATP balance within the cell (Erdrich et al ., 2014 ). To counteract such an imbalance, cyclic electron transfer (CET) can be downregulated. However, when only linear electron transfer is operating, an increasing adenylate energy charge together with an increasing redox ratio in terms of NADP + /NADPH may cause Calvin‐Benson‐Bassham (CBB) cycle limitation (by NADPH) and/or inhibition. The latter may be mediated by regulators like thioredoxin (Mallén‐Ponce et al ., 2021 ) or small proteins like CP12 (Brandenburg and Klähn, 2020 ; McFarlane et al ., 2019 ), which are redox‐regulated, with the redox state serving as a proxy for light availability (Brandenburg and Klähn, 2020 ). As a consequence, the CBB cycle activity may be significantly decreased upon NADPH withdrawal, in turn reinforcing the NADPH:ATP imbalance due to lacking ATP sink (Cano et al ., 2018 ). This then also may harm the photosynthetic machinery, for example, its regeneration and oxidative stress management, finally leading to a decrease in light reaction rates and thus fostering NADPH limitation (Brandenburg and Klähn, 2020 ; Mallén‐Ponce et al ., 2021 ; McFarlane et al ., 2019 ; Ogawa et al ., 2021 ). Thus, the NADPH:ATP imbalance caused by extensive NADPH withdrawal may shut down the photosynthetic electron supply, resulting in a metabolic stalemate. One possible way to escape it has been lately reported: It was shown that co‐expression of an ATP‐sink together with an electron sink could substantially benefit the latter one (Santos‐Merino et al ., 2021 ). Following this hypothesis, a moderate burden would avoid or lead to a weak NADPH:ATP imbalance, which can be endured by the cell without drastic measures, enabling a stable biotransformation. Other strategies to stabilize instabilities encountered with cyanobacteria have recently been reviewed by Guillaume and dos Santos, with the use of genome‐scale metabolic models as a promising option to mitigate instability and design stable production strains (Guillaume and Branco Dos Santos, 2023 ). Further research is needed to elucidate the occurrence and effects of NADPH:ATP imbalances. The synergy of biochemical and biocatalyst engineering, including the involvement of a reaction cascade, enabled a stable bioconversion in Synechocystis for up to 48 h, reaching a final titre of 23.50 ± 0.84 m m 6‐HA, unprecedented for light‐driven oxygenase reactions. Besides the excellent specificity ( Y \n P/S = 0.96 mol/mol), the high biocatalyst yield of 3.71 ± 0.21 g 6‐HA /g CDW illustrates the benefit of a high process stability. Consequently, the presented enzyme cascade outcompetes described photosynthesis‐driven oxygenase‐based systems regarding both volumetric productivity and final product titre and, thus, holds great promise for future applications of Synechocystis as a host for photo‐biotechnological applications. However, space–time‐yields and final titers still need substantial improvements to make phototrophic microbes commercially applicable as sustainable cell factories. Metabolic engineering, for example, to balance energy (ATP) and redox demands (NADPH), will play an important role in further increase rates of photosynthesis‐driven biocatalysis in Synechocystis (Toepel et al ., 2023 ). A main challenge for efficient scale‐up is the typically low cell density in phototrophic cultivation systems, being limited by light input and penetration. Recent developments like internal illumination (Hobisch et al ., 2021 ) and capillary biofilm reactors (Hoschek et al ., 2019 ) constitute highly attractive concepts to meet this challenge. The estimation of quantum and light usage efficiencies allows for a quantitative assessment of photosynthesis‐driven biocatalysis: Following the calculations for the high C‐one feed regime (Table 1 ), a remarkable share of 40% of intracellularly available reductant was allocated to the biotransformation during the first hour. As discussed above, this high share could not be sustained in the long run. In contrast, the activity limited by a moderate substrate feed, consuming 16.5% of cellular reductant, could be sustained for 30 h, with substantial activity being maintained for at least 48 h without compromising growth. In fact, the biotransformation could be supplied with reduction equivalents in addition to deducting reduction equivalents from growth. Consequently, the biotransformation increased the overall light conversion efficiency (3.6% vs. 2.5%, Table 1 ). Hence, the biotransformation appeared to be sustained with cellular energy that otherwise would be dissipated and lost from a biotechnological point of view. The sustained quantum efficiency of product formation and the product yield on light were 2.6% and 0.7%, respectively, representing considerable values, given that both strain and reactor format is far from being optimized for efficient light usage. The overall light conversion efficiency (3.6%) is remarkable in view of the fact that the theoretical maximum conversion efficiency of photosynthesis is debated to lie between 4.5% (Barber, 2009 ) and 10% (Bolton, 1977 ) and real efficiencies of up to 2% (Barber, 2009 ; Jakob et al ., 2007 ; Melis, 2009 ). Strain and reactor engineering augur well to leverage the full potential of photosynthesis‐driven redox biocatalysis. The enzyme cascade described in this study constitutes an excellent model system to investigate physiological responses to a strong electron sink, fulfilling two important criteria: (i) It is a tunable reaction, for example, by substrate supply and/or expression strength and (ii) it allows for a stable conversion for not only hours, but even days. As such it is ideal to study the regulatory processes, for example, regarding the NADPH:ATP balance, with both omics techniques to investigate effects on the transcript, protein and metabolome levels and state‐of‐the‐art fluorometry methods to examine photosynthetic apparatus operation. Especially in the light of envisioned bulk chemical production like light‐driven H 2 formation, it will be essential to follow up on the question, which share of reductant can be withdrawn from cyanobacterial metabolism. The data presented in this study for fed‐batch biotransformation give stimulating insights, but cannot be regarded as a quantitative assessment of the photosynthetic capacity, which needs to be investigated in more detail in future."
} | 4,400 |
20632001 | null | s2 | 4,168 | {
"abstract": "Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p < 0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development."
} | 421 |
26609321 | PMC4659176 | pmc | 4,171 | {
"abstract": "Background The surplus of glycerol has increased remarkably as a main byproduct during the biofuel’s production. Exploiting an alternative route for glycerol utilization is significantly important for sustainability of biofuels. Results A novel biocatalyst that could be prepared from glycerol for producing 2-oxo-carboxylates was developed. First, Pseudomonas putida KT2440 was reconstructed by deleting lldR to develop a mutant expressing the NAD-independent lactate dehydrogenases (iLDHs) constitutively. Then, the Vitreoscilla hemoglobin (VHb) was heterologously expressed to further improve the biotransformation activity. The reconstructed strain, P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , exhibited high activities of iLDHs when cultured with glycerol as the carbon source. This cost-effective biocatalyst could efficiently produce pyruvate and 2-oxobutyrate from dl -lactate and dl -2-hydroxybutyrate with high molar conversion rates of 91.9 and 99.8 %, respectively. Conclusions The process would not only be a promising alternative for the production of 2-oxo-carboxylates, but also be an example for preparation of efficient biocatalysts for the value-added utilization of glycerol.",
"conclusion": "Conclusions Taking the 2-oxo-carboxylates production as an example, we developed a process of using biofuel’s byproduct glycerol for biocatalyst preparation. After deleting the LldR and heterologously expressing VHb in P. putida KT2440, the recombinant P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb with high iLDHs activities was cost-effectively prepared from glycerol. Using the whole cells as biocatalyst, 90.9 mM pyruvate and 99.3 mM 2-OBA were produced in 6 h from 100 mM dl -lactate and dl -2-HBA, respectively. The process demonstrated an option for effective utilization of the low-cost and renewable substrates through recombining the metabolic networks, based on the regulation mechanism, with the goal of producing the high-value chemicals.",
"introduction": "Effect of VHb introduction in whole-cell biocatalysis It was also investigated if the introduction of VHb could affect the whole cells biocatalysis activities. The biocatalysis reactions were conducted at 30 °C in phosphate buffer (pH 7.4) for 6 h, with 10.5 g dry cell weight (DCW)/L of P. putida KT2440, P. putida KT2440/pBSPPc Gm - vgb , P. putida KT2440 (Δ lldR ) and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , which cultured with glycerol, as the biocatalysts, respectively. l -Lactate, d -lactate, and racemic lactate at 100 mM were used as the substrates. The biocatalysis reactions were carried out in the presence of 30 mM ethylenediaminetetraacetic acid (EDTA), which could remove bivalent ions necessary for 2-keto-acid decarboxylase-catalyzed reactions [ 45 , 46 ], and then could block the degradation of 2-oxo-carboxylates. As shown in Fig. 2 d, via 6 h biotransformation, no pyruvate production was detected when either P. putida KT2440 or P. putida KT2440/pBSPPc Gm - vgb was used, which was in correspondence to the result of the activities assays of iLDHs of these two strains. However, both P. putida KT2440 (Δ lldR ) and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb have the ability to oxidize the two enantiomers of lactate. For P. putida KT2440 (Δ lldR ), the oxidation rates toward l -lactate, d -lactate and dl -lactate to pyruvate were 0.38 mmol/g DCW h, 0.34 mmol/g DCW h and 0.51 mmol/g DCW h, respectively. And for P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , the oxidation rates toward these three kinds of lactate have remarkably increased to 0.77 mmol/g DCW h, 0.60 mmol/g DCW h and 1.00 mmol/g DCW h, respectively (Fig. 2 d). As expected, the reconstructed strain with heterologously expressed VHb exhibited about twofold higher biotransformation activities than the strain without VHb expression. These results revealed that the introduction of VHb into the recombinant P. putida KT2440 (Δ lldR ) indeed significantly enhanced the whole cells biocatalysis activities of lactate oxidation to produce pyruvate. Furthermore, the pyruvate production rates from dl -lactate were significantly higher than which from either l -lactate or d -lactate alone (Fig. 2 d). l -iLDH and d -iLDH catalyze the oxidation of l -lactate and d -lactate, respectively. The higher biotransformation activity toward dl -lactate might be due to the fact that both isomers in dl -lactate would be simultaneously oxidized by l -iLDH and d -iLDH in these recombinant strains. Compared with optical pure lactate, the low price and large sources of racemic lactate make it become a more cost-effective substrate to produce pyruvate. Based on the results above, the P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , a recombinant strain with constitutive iLDHs and heterologously expressed VHb, has the potential to efficiently produce 2-oxo-carboxylates from 2-hydroxy-carboxylates.",
"discussion": "Results and discussion Regulatory networks of glycerol and lactate metabolism in P. putida KT2440 Owing to its versatile metabolic activities, P. putida KT2440 can use various organics as carbon and energy sources, which make this strain an ideal industrial microorganism used for biotransformation and biodegradation [ 21 ]. However, most of the metabolism networks are subject to the strict regulation. Take glycerol and lactate as examples, both of these two compounds can be used as carbon and energy sources for P. putida KT2440 [ 9 , 22 ]. During the glycerol utilization process, the specific enzymes related to the glycerol metabolism, including GlpF (a glycerol transporter encoded by glpF ), GlpK (a glycerol kinase encoded by glpK ), and GlpD (a glycerol-3-phosphate dehydrogenase encoded by glpD ), will be generally induced [ 9 ]. The expressions of these genes are regulated by GlpR, a DeoR family transcriptional regulator, which is encoded by glpR (PP1074) [ 9 , 23 , 24 ]. The expression of glpR is not affected by glycerol [ 9 ]. The primary structure and N-terminal helix-turn-helix (HTH) DNA-binding motif of GlpR in Escherichia coli K12 have already been identified [ 25 ]. Moreover, previous research has shown that the GlpR of E. coli K12 is a tetramer under native conditions [ 26 ]. Since the GlpR from E. coli K12 and P. putida KT2440 share the high consensus positions (71.0 %) and identity positions (54.8 %), we conjectured that the GlpR from P. putida KT2440 might be a tetramer and possess N-terminal HTH DNA-binding motifs as well. The structure of C-terminal effector-binding domain of DeoR from Bacillus subtilis has been determined [ 27 ]. In view of the fact that the GlpR belongs to the DeoR family transcriptional regulator [ 25 ], the GlpR might also possess the similar C-terminal effector-binding domains. Based on these backgrounds mentioned above, the hypothetic schematics of the regulatory networks of the GlpR-depended glycerol metabolism in P. putida KT2440 are shown in Fig. 1 . As shown in Fig. 1 a, when P. putida KT2440 is cultured with the glycerol as a carbon source, the glycerol from the extracellular environment will be transported into cytoplasm (mediated by GlpF) [ 24 ]. The sn-glycerol-3-P (G3P) produced by the substrate phosphorylation of glycerol (mediated by GlpK, the glycerol kinase) will be generated via the effector-independent expression of glp genes, which occurs in a low-probability stochastic way [ 24 ]. And then the repression of glp genes mediated by GlpR will be derepressed by the increasing intracellular G3P [ 24 ]. As an effector for GlpR, G3P can bind to the effector-binding domains of GlpR, and then will prevent the HTH DNA-binding domains of GlpR from binding to the DNA operator sequence of glp regulon [ 24 , 25 , 27 ]. On the contrary, the free tetrameric assembly of GlpR will tightly bind to the DNA operator sequence and inhibit the expressions of glp genes when the strain cultured without glycerol (Fig. 1 b) [ 24 , 27 ]. Fig. 1 The hypothetic schematics of regulatory networks of glycerol and lactate metabolism in P. putida KT2440, and the construction of P. putida KT2440 (Δ lldR ). a The derepression of the glp genes expression is occurring when the strain cultured in MSM with glycerol as carbon source. On the other hand, the expressions of lldPDE genes are repressed by the LldR, without the lactate as inducer. b The derepression of the lldPDE genes expression is occurring when the strain cultured in MSM with dl -lactate as carbon source. On the contrary, the free tetrameric assembly of GlpR will tightly bind to the DNA operator sequence with the DNA-binding domains, and inhibit the expressions of glp genes. c If the lldR gene is deleted, the repression of the lldPDE genes expression will be damaged, owing to the absence of the functional LldR. And the lldPDE genes will still fully express, even without lactate as the inducer. d Diagram illustrating the disruption of the lldR mediated by homologous double crossover. e Analysis of PCR fragments to confirm lldR disruption. Lane M molecular mass standard (λDNA/HindIII); lane 1 product amplified with P. putida KT2440 genomic DNA as the template; lane 2 product amplified with water as the template (negative control); lane 3 product amplified with P. putida KT2440 (Δ lldR ) genomic DNA as the template. The PCRs were performed with primers lldR k.f and lldR k.r The similar regulation network also exists in the lactate metabolism of P. putida KT2440. The lldPDE operon, which is responsible for lactate utilization in most Pseudomonas strains including P. putida KT2440, has been studied in previous reports [ 19 , 28 – 31 ]. The lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an l -iLDH), and lldE (encoding a d -iLDH) [ 19 , 22 ]. The l -iLDH and d -iLDH are mainly responsible for the oxidation of l -lactate and d -lactate to pyruvate, respectively [ 19 , 28 , 31 ]. However, the expressions of lldD and lldE are both repressed by the regulator LldR which is encoded by upstream adjacent gene lldR [ 19 ]. The previous study reported that the LldR from Corynebacterium glutamicum is a homodimer assembled by domain swapping [ 32 ]. The N-domain of this regulator contains a typical winged HTH (WHTH) DNA-binding domain [ 32 , 33 ]. And the C-terminal domain is assumed to play the ligand-binding role [ 32 ]. As the primary structure predicted by NCBI, the monomer of LldR from P. putida KT2440 comprises an N-terminal WHTH DNA-binding and a C-terminal ligand-binding domain, which is coincident with the typical region features of many members of the GntR family [ 34 ]. The hypothetic schematics of the regulatory networks of the LldR-mediated lactate metabolism in P. putida KT2440 have also been shown in Fig. 1 . As shown in Fig. 1 a, while the strain cultured in the medium without lactate, the free LldR homodimer can bind to the promoter region of the lldPDE operon with the WHTH DNA-binding domains, and will inhibit the expressions of lldPDE genes downstream. However, the derepression of LldR to the lldPDE genes is occurring when lactate exists in the growing environment of the strain. As an effector of LldR, the lactate can bind to the C-terminal ligand-binding domains. Then, the effector-bound LldR will lose the ability of binding to the DNA promoter sequence of lldPDE regulon (Fig. 1 b). Reconstruction of P. putida KT2440 lldR deletion mutant As mentioned before, the presence of lactate is necessary for the expressions of l -iLDH and d -iLDH [ 19 , 31 , 35 ]. As a result, the hyperosmotic medium caused by high concentration of lactate becomes a major limitation for high-density culture, and the indispensable lactate addition raises the cost of biocatalysts preparation. Considering the versatile applications of P. putida KT2440 in biocatalysis, it is rather desirable to prepare efficient biocatalyst from a more cost-effective substrate, such as glycerol, with the required enzymes iLDHs. To achieve this goal, the regulatory network of lactate utilization was reconstructed. After deleting the lldR gene, it seems likely that the regulatory network would be broken down because of the absence of the functional LldR. Therefore, when cells incubated with the glycerol as the sole carbon source, the lldPDE genes would still fully express, even without the induction of lactate (Fig. 1 c). To explore this possibility, we disrupted the lldR gene which encodes negative regulator LldR in P. putida KT2440. The suicide plasmid pK18 mobsacB which mediated the homologous recombination was used for deleting the lldR gene (Fig. 1 d) [ 36 ]. The disruption of the gene lldR was verified by PCR (Fig. 1 e). The result strain is named P. putida KT2440 (Δ lldR ). Effect of inactivation of lldR on iLDHs expression The wild-type P. putida KT2440 and P. putida KT2440 (Δ lldR ) were cultured in 500-mL baffled shake flasks each containing 100 mL minimal salt medium (MSM) [ 31 ] supplied with 5 g/L dl -lactate or glycerol as the carbon source. And 1 mM octanoate, as the co-feeder, was added to the MSM with glycerol to shorten lag phase [ 23 ]. To investigate the effect of inactivation of lldR on iLDHs expression, the activities of l -iLDH and d -iLDH in crude cell extracts of P. putida KT2440 and P. putida KT2440 (Δ lldR ) were assayed, with 2,6-dichloroindophenol (DCIP) as the artificial electron acceptor and 20 mM l - or d -lactate as the electron donor. As shown in Table 1 , when the P. putida KT2440 was cultured in the medium with dl -lactate as the sole carbon source, the enzymes activities of l -iLDH and d -iLDH were 161.4 nmol/min mg protein and 332.9 nmol/min mg protein, respectively. However, neither l -iLDH nor d -iLDH activity was detectable in P. putida KT2440 when cultured with glycerol. Comparatively, when the P. putida KT2440 (Δ lldR ) was incubated in the MSM with dl -lactate as the carbon source, the activity of l -iLDH was 220.9 nmol/min mg protein and d -iLDH was 471.5 nmol/min mg protein. While incubated in the MSM containing the glycerol as the carbon source, this Δ lldR mutant also exhibited high activities of iLDHs, 348.2 nmol/min mg protein and 771.0 nmol/min mg protein for l -iLDH and d -iLDH, respectively. These results revealed that the repression effects of LldR on l -iLDH and d -iLDH expressions were removed by the disruption of lldR gene. When cultured with glycerol, the iLDHs activities of the P. putida KT2440 (Δ lldR ) were not impaired, compared with that cultured with dl -lactate. Therefore, the P. putida KT2440 (Δ lldR ), in which l -iLDH and d -iLDH are expressed constitutively, has the potential to efficiently produce pyruvate from lactate with glycerol as the cost-effective culture substrate. Table 1 Activities of iLDH s in crude cell extracts of P. putida KT2440 and P. putida . KT2440 (∆ lldR ) cultured with different growth substrates Growth substrate Strain Enzyme activity (nmol/min mg protein) a \n \n l -iLDH \n d -iLDH \n dl -Lactate \n P. putida KT2440 161.4 ± 4.6 332.9 ± 4.3 \n P. putida KT2440 (∆ lldR ) 220.9 ± 7.0 471.5 ± 3.5 Glycerol \n P. putida KT2440 ND ND \n P. putida KT2440 (∆ lldR ) 348.2 ± 11.2 771.0 ± 9.0 \n ND not detected \n a Activities of d -iLDH and l -iLDH were examined with 20 mM d -lactate or 20 mM l -lactate. DCIP was used as the electron acceptor. Results are mean ± SD of three parallel replicates Vitreoscilla hemoglobin (VHb) enhances the lactate oxidation It has been revealed in a previous study that iLDHs from P. stutzeri SDM could not oxidize lactate with oxygen as the directly electron acceptor [ 37 ]. The lldPDE operon organization is similar in P. putida KT2440 and P. stutzeri SDM, and the lactate utilization genes between these two strains show strikingly high homology [ 19 , 29 , 38 ]. It is inferred that the electron produced in the lactate oxidation process might terminally transfer to the oxygen, a final electron acceptor, through the electron transport chain in P. putida KT2440, as well as in P. stutzeri SDM [ 37 ]. Vitreoscilla hemoglobin (VHb) is a soluble homodimeric globin encoded by vgb , a 438 bp gene discovered in Vitreoscilla sp. [ 39 ]. It is the first bacterial hemoglobin whose structure and function have been well characterized [ 39 , 40 ]. Since the gene ( vgb ) encoding VHb has been cloned [ 41 , 42 ], its heterologous expression has become an engineering strategy widely used to increase production of a diverse of bioproducts and facilitate the bioremediation [ 43 ]. In this study, the vgb gene was amplified with the primers vgb.f and vgb.r from the vector pET28b-RgDAAO-VHb. Then, the 438 bp vgb fragment was ligated to Hind III and BamH I double-digested pBSPPc Gm , a broad-host-range constitutive vector containing a P c promoter [ 44 ], to produce pBSPPc Gm - vgb (Fig. 2 a). As shown in Fig. 2 b, the vgb gene has been successfully cloned and inserted into the pBSPPc Gm with the corresponding restriction enzyme sites, obtaining the pBSPPc Gm - vgb . The vector was transferred into P. putida KT2440 and P. putida KT2440 (Δ lldR ) by electroporation to produce P. putida KT2440/pBSPPc Gm - vgb and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , respectively. Fig. 2 Construction of the vgb expressing vector and comparisons of iLDHs activities and biotransformation rates toward pyruvate production between different P. putida KT2440 strains. a Construction of recombinant vector pBSPPc Gm - vgb . vgb , the gene encoding VHb. pBSPPc Gm , a broad-host-range constitutive vector containing a P \n c promoter. The vgb gene was inserted into the pBSPPc Gm - vgb in the corresponding sites, to generate the plasmid pBSPPc Gm - vgb . b Verification of pBSPPc Gm - vgb. Lane M molecular mass standard (λDNA/HindIII); lane 1 product amplified with pET28b-RgDAAO-VHb as the template; lane 2 double enzymes digestion ( HindIII and BamHI ) of recombinant vector pBSPPc Gm - vgb . c Activities of l -iLDH ( violet bars ) and d -iLDH ( light magenta bars ) in crude cell extracts of P. putida KT2440 and its derivatives were examined with DCIP as the artificial electron acceptor and 20 mM l - or d -lactate as the electron donor. Results are mean ± SD of three parallel replicates. d The biotransformation rates of pyruvate production by whole cells of different P. putida KT2440 strains. The biotransformations were conducted with the whole cells of P. putida KT2440, P. putida KT2440/pBSPPc Gm - vgb , P. putida KT2440 (Δ lldR ) and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb with 100 mM l -lactate ( violet bars ), 100 mM d -lactate ( light cyan bars ) and 100 mM dl -lactate ( blue bars ) as the substrates. The concentrations of the pyruvate were measured by HPLC. Results are mean ± SD of three parallel replicates To explore the effect of introduction of VHb in lactate oxidation, the l -iLDH and d -iLDH activities in the crude cell extracts of P. putida KT2440, P. putida KT2440 (Δ lldR ), P. putida KT2440/pBSPPc Gm - vgb and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , cultured with glycerol, were assayed. As the results shown in Fig. 2 c, the introduction of VHb did not increase the activities of iLDHs in P. putida KT2440 cultured with the glycerol as carbon source. The enzymes activities of l -iLDH and d -iLDH in P. putida KT2440 (Δ lldR ) were 273.0 nmol/min mg protein and 595.0 nmol/min mg protein, respectively. However, compared with P. putida KT2440 (Δ lldR ), both the activities of l -iLDH and d -iLDH in P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb significantly increased to 520.7 nmol/min mg protein and 1063.9 nmol/min mg protein, respectively, almost twofold higher than those in P. putida KT2440 (Δ lldR ). Effect of VHb introduction in whole-cell biocatalysis It was also investigated if the introduction of VHb could affect the whole cells biocatalysis activities. The biocatalysis reactions were conducted at 30 °C in phosphate buffer (pH 7.4) for 6 h, with 10.5 g dry cell weight (DCW)/L of P. putida KT2440, P. putida KT2440/pBSPPc Gm - vgb , P. putida KT2440 (Δ lldR ) and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , which cultured with glycerol, as the biocatalysts, respectively. l -Lactate, d -lactate, and racemic lactate at 100 mM were used as the substrates. The biocatalysis reactions were carried out in the presence of 30 mM ethylenediaminetetraacetic acid (EDTA), which could remove bivalent ions necessary for 2-keto-acid decarboxylase-catalyzed reactions [ 45 , 46 ], and then could block the degradation of 2-oxo-carboxylates. As shown in Fig. 2 d, via 6 h biotransformation, no pyruvate production was detected when either P. putida KT2440 or P. putida KT2440/pBSPPc Gm - vgb was used, which was in correspondence to the result of the activities assays of iLDHs of these two strains. However, both P. putida KT2440 (Δ lldR ) and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb have the ability to oxidize the two enantiomers of lactate. For P. putida KT2440 (Δ lldR ), the oxidation rates toward l -lactate, d -lactate and dl -lactate to pyruvate were 0.38 mmol/g DCW h, 0.34 mmol/g DCW h and 0.51 mmol/g DCW h, respectively. And for P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , the oxidation rates toward these three kinds of lactate have remarkably increased to 0.77 mmol/g DCW h, 0.60 mmol/g DCW h and 1.00 mmol/g DCW h, respectively (Fig. 2 d). As expected, the reconstructed strain with heterologously expressed VHb exhibited about twofold higher biotransformation activities than the strain without VHb expression. These results revealed that the introduction of VHb into the recombinant P. putida KT2440 (Δ lldR ) indeed significantly enhanced the whole cells biocatalysis activities of lactate oxidation to produce pyruvate. Furthermore, the pyruvate production rates from dl -lactate were significantly higher than which from either l -lactate or d -lactate alone (Fig. 2 d). l -iLDH and d -iLDH catalyze the oxidation of l -lactate and d -lactate, respectively. The higher biotransformation activity toward dl -lactate might be due to the fact that both isomers in dl -lactate would be simultaneously oxidized by l -iLDH and d -iLDH in these recombinant strains. Compared with optical pure lactate, the low price and large sources of racemic lactate make it become a more cost-effective substrate to produce pyruvate. Based on the results above, the P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , a recombinant strain with constitutive iLDHs and heterologously expressed VHb, has the potential to efficiently produce 2-oxo-carboxylates from 2-hydroxy-carboxylates. Pyruvate and 2-OBA production through whole-cell biocatalysis The oxidation of two most important members of 2-hydroxy-carboxylates, lactate and 2-hydroxybutyrate (2-HBA) that are catalyzed by iLDHs, have been studied in previous studies [ 37 , 47 ]. In this study, the biocatalytic oxidation of racemic lactate (100 mM) and 2-HBA (100 mM) were carried out, with 10.5 g DCW/L of whole cells of P. putida KT2440, P. putida KT2440/pBSPPc Gm - vgb , P. putida KT2440 (Δ lldR ) and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , which were prepared from glycerol, as the biocatalysts, respectively. The biocatalysis reactions were conducted at 30 °C in phosphate buffer (pH 7.4) with the presence of 30 mM EDTA. As shown in Tables 2 and 3 , the racemic lactate (100 mM) and 2-HBA (100 mM) were completely oxidized into pyruvate and 2-OBA, via 6-h bioconversion with P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb as the biocatalyst. The yields of pyruvate and 2-OBA with P. putida KT2440 (Δ lldR ) were 50.9 and 74.7 %, respectively. However, the yields of these two productions with P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb were 91.9 and 99.8 %, respectively, about 1.8-fold and 1.3-fold higher than which catalyzed by P. putida KT2440 (Δ lldR ). The final concentrations of pyruvate and 2-OBA produced by P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb were 90.9 mM and 99.3 mM, respectively. Table 2 Comparison of pyruvate productions by whole cells of P. putida KT2440, P. putida /pBSPPc Gm - vgb , P. putida KT2440 (Δ lldR ), and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb \n Strain Pyruvate (mM) Yield (%) a \n Productivity (mmol/g DCW h) KT2440 0 0 0 KT2440/pBSPPc Gm - vgb \n 0 0 0 KT2440 (Δ lldR ) 50.60 ± 0.38 50.9 0.81 ± 0.005 KT2440 (Δ lldR )/pBSPPc Gm - vgb \n 90.85 ± 0.75 91.9 1.43 ± 0.002 The initial dl -lactate concentration was 100 mM. The biocatalysis reactions were conducted at 30 °C in phosphate buffer (pH 7.4) for 6 h with 10.5 g DCW/L of biocatalysts prepared from glycerol. Results are mean ± SD of three parallel replicates \n a The yields of pyruvate were calculated based on the actual initial concentrations of dl -lactate measured by HPLC Table 3 Comparison of 2-OBA productions by whole cells of P. putida KT2440, P. putida KT2440/pBSPPc Gm - vgb , P. putida KT2440 (Δ lldR ), and P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb \n Strain 2-OBA (mM) Yield (%) a \n Productivity (mmol/g DCW h) KT2440 0 0 0 KT2440/pBSPPc Gm - vgb \n 0 0 0 KT2440 (Δ lldR ) 75.52 ± 1.59 74.7 1.20 ± 0.03 KT2440 (Δ lldR )/pBSPPc Gm - vgb \n 99.31 ± 1.45 99.8 1.58 ± 0.02 The initial dl -2-HBA concentration was 100 mM. The biocatalysis reactions were conducted at 30 °C in phosphate buffer (pH 7.4) for 6 h with 10.5 g DCW/L of biocatalysts prepared from glycerol. Results are mean ± SD of three parallel replicates \n a The yields of 2-OBA were calculated based on the actual initial concentrations of dl -2-HBA measured by HPLC Many researches have focused on the conversions of the inexpensive glycerol into high-value products, such as fine chemicals [ 6 ] and biodiesels [ 16 ], via the microbial fermentation. Furthermore, glycerol can also be used as a carbon source and energy source to support the growth of many industrial microorganisms [ 8 ]. The development of biotechnology of glycerol utilization processes will allow the biofuel industry to be more competitive. Compared with lactate, glycerol is a more cost-effective green substrate suitable for preparation of the biocatalyst containing the iLDHs. In 2006 and 2007, the spot price of the heat-stable lactic acid, as posted in the Chemical Marketing Reporter, was about $0.70 per pound [ 48 ]. In contrast, the prices of refined glycerol and crude glycerol were approximately $0.30 and $0.050 per pound, respectively, obviously cheaper than the lactic acid [ 49 ]. Furthermore, as a byproduct of biofuels, large amounts of glycerol have become a waste stream. Making good use of this waste stream would not only lower the production costs, but also contribute to the sustainable development of the biofuels industry. After reconstructing the lactate utilization regulatory network by deleting the LldR regulator in P. putida KT2440, the Δ lldR mutant exhibited the outstanding constitutive iLDHs activities when cultured with glycerol as the carbon source (Fig. 3 ). On the other hand, a constitutive vector with high expression strengths and broad host ranges was used for the introduction of VHb, which can enhance the lactate oxidation activities and the yields of the 2-oxo-carboxylates significantly (Fig. 3 ). This beneficial effect of VHb heterologous expression may be the result of binding more oxygen and delivering to the respiratory chain [ 43 ]. It is also feasible to apply this VHb introduction technology in other aerobic biotransformations to increase production efficiency. Fig. 3 The optimization process of the biocatalysts prepared with glycerol Pyruvate and 2-OBA are two important platform compounds which have been widely applied in the chemical, drug, and food industries [ 47 , 50 ]. Pyruvate has been used as a weight-control dietary supplement, a supplemental nutrient, and an antioxidant which can protect the brain and other tissues from the oxidative stress [ 50 – 53 ]. 2-OBA is an important intermediate widely used in biosynthesis of l -isoleucine, d -2-hydroxybutyrate and 1-propanol [ 54 – 56 ]. Furthermore, 2-OBA could be bioconverted into a non-natural amino acid l -homoalanine, which is a key chiral precursor for production of levetiracetam, brivaracetam, and ethambutol [ 57 , 58 ]. Owing to the mild reaction conditions, high substrate conversion efficiency and simple compositions of the reaction mixture which is contributed to the convenience of recovery, whole cell catalysis becomes the preferred method for pyruvate and 2-OBA production [ 47 , 50 ]. For example, Pseudomonas stutzeri SDM, which contains inducible iLDHs, has been reported to have good ability to produce pyruvate and 2-OBA from lactate and 2-HBA as the substrates, respectively [ 37 , 47 ]. Whole cells of P. stutzeri SDM with iLDHs must be prepared with lactate as the carbon source. Although the concentrations of pyruvate and 2-OBA reported here were lower than these previous reports, this work disclosed a novel biocatalyst which could be prepared with glycerol as a more cost-effective substrate."
} | 7,325 |
31477777 | PMC6718607 | pmc | 4,172 | {
"abstract": "Feeding Bombyx mori larvae with chemically-modified diets affects the structure and properties of the resulted silk. Herein, we provide a road map for the use of silkworms as a factory to produce semiconducting/metallic natural silk that can be used in many technological applications such as supercapacitor electrodes. The silkworms were fed with four different types of chemicals; carbon material (graphite), sulfide (MoS 2 ), oxide (TiO 2 nanotubes), and a mixture of reactive chemicals (KMnO 4 /MnCl 2 ). All the fed materials were successfully integrated into the resulted silk. The capacitive performance of the resulted silk was evaluated as self-standing fabric electrodes as well as on glassy carbon substrates. The self-standing silk and the silk@glassy carbon substrate showed a great enhancement in the capacitive performance over that of the unmodified counterparts. The specific capacitance of the self-standing blank silk negative and positive electrodes was enhanced 4 and 5 folds at 10 mV/s, respectively upon the modification with KMnO 4 /MnCl 2 compared to that of the plain silk electrodes.",
"conclusion": "Conclusions We demonstrate the ability to fabricate functionalized natural silk fibers by feeding the silkworms with the material of interest. Specifically, this work highlights the possibility of using natural silk fibers as supercapacitor electrodes upon feeding the worms with high capacitive materials such as graphite, MoS 2 , TiO 2 , and KMnO 4 /MnCl 2 . The study showed that the fed material did not greatly affect the crystallinity of the silk fibroin and all the added materials enhanced the capacitance performance and the thermal stability of the silk fibers. It was observed that both S/B and S/Mn contained more β-sheet silk, have close thermal stability, and both acted better as negative electrodes. The study proved that natural silk can be tuned for use in energy storage devices.",
"introduction": "Introduction Metals and semiconductors are the backbone of our modern industry. Therefore, there is a continuous need to develop new methods and technologies to produce such essential materials with the desired characteristics at low cost. Of special interest, enormous efforts have been devoted to developing flexible wearable devices. Those wearable devices are usually made of synthetic nanofibers. However, one of the cheapest and commonly used fibers is the natural silk (NS) 1 , 2 , which has been used, through many decades, as fabric for many applications such as biodegradable medical implants, durable protective fabrics, and eco-friendly wearable electronics 3 – 5 . NS consists mainly of a polymerized protein known as fibroin covered with a glue-like material named sericin 6 . It is fabricated through the organisms of silkworms from a liquid combination of polymers at room temperature, resulting in a silk that is insoluble in water 3 , 7 . The fibroin of the Bombyx mori larvae is a semi-crystalline biopolymer consisting of glycine, alanine and serine 8 . However, the as-produced spun silks are usually treated with additives to make them functional, which adds to the cost and requires tedious optimization. A promising approach to overcome such obstacles can occur through additives to the food of the silkworms (usually mulberry leaves) 3 , 8 . Feeding the worms with special chemical materials, which can be incorporated in the glands of the worms and mix with the fibroin liquid, is expected to result in a modified-silk composite that comprises the properties of both NS and the incorporated materials 6 – 9 . The fact that NS radiates heat more than it absorbs and self-cool, makes it a good candidate for electronic applications 10 . Feeding Bombyx mori larvae with nanostructured materials such as CNTs 7 , 8 , graphene 7 , TiO 2 9 , 11 and other metal oxides 6 have been investigated in recent reports. The feeding process proved that Bombyx mori larvae can intake nanostructured materials, which affect the crystallinity of the resulting silk. Feeding the worms with TiO 2 was also proved to be nontoxic 11 and even used with bacteria to enhance energy harvesting devices 12 . However, most of the previous reports were limited to the investigation of the mechanical and photonic properties of such modified silk 7 , 8 . Tailoring the properties of the NS to be used in electronic devices, energy generation, and energy storage devices is yet to be reported. Of special interest, flexible supercapacitors are emerging as promising platforms for energy storage 13 – 15 . Herein, we demonstrate the ability to modify the structure and supercapacitive behavior of NS by feeding the Bombyx mori larvae with four different types of materials (graphite, TiO 2 nanotubes, MoS 2 , and KMnO 4 /MnCl 2 ) for use as supercapacitor electrodes. The study shows that modification of the NS enhanced its capacitive behavior, paving the way for their use in flexible supercapacitor applications.",
"discussion": "Results and Discussion Effect of the feeding process All the studied silkworms started the feeding on their 5 th instar and they did not reject the food. It was observed that the larvae fed with MoS 2 were eating more than usual while the ones fed with KMnO 4 /MnCl 2 were eating in a lower rate than usual. The larvae fed with graphite and TiO 2 did not show any unusual behavior in the feeding process. While the Cocoons of the blank fed larvae were of homogeneous size and white color, the chemically-modified ones showed a non-homogenous size and off-white color. After degumming, all the fabricated fibers were of a clear white color. The resulted silk was given the names S/B, S/G, S/TiO 2 , S/MoS 2 and S/Mn for the blank silk, the graphite modified silk, the TiO 2 modified silk, the MoS 2 modified silk and the KMnO 4 /MnCl 2 , respectively. Structure of the resulted silk The morphology of the silk fibers was investigated using FESEM imaging as shown in Fig. 2 . Note that the thickness of the fabricated fibers is independent of the type of the chemical additive, having diameters ranging from 9 to 16 µm, in agreement with previous reports 8 , 20 . The fed materials appeared as debris on the surface of the fibers and/or within their internal fibroins. While the S/B fibers showed a trigonal shaped cross-section as presented in the inset of Fig. 2 , the S/G and S/TiO 2 showed an oval-shaped cross-section with the additives clearly appearing on the surface of the fibers. However, the S/MoS 2 and S/Mn showed a flattened oval cross-section and the fibers were more flat than usual, which may suggest that the additives (MoS 2 , Mn) were interfered with the fiber materials and reconstructed its protein structure 8 . The elemental composition of the fibers was studied using the EDS technique and the results are presented in Table 1 . The resulted composition showed that the added material did not exceed 0.03 at% of the total atoms in the fiber, which is an accepted ratio due to the low concentration (0.5 wt% suspension) used in the diet. The S/B and S/G did not vary greatly due to the fact that graphite is only made of carbon atoms. However, the S/MoS 2 analysis showed 0.03 at% of Mo and 0.05 atom% of S. the S/TiO 2 showed a Ti composition of 0.03 at% and the S/Mn showed 0.02 at% of Mn and 0.01 at% of K “from the added KMnO 4 ”, with no signal for Cl atoms at different positions of the S/Mn fibers indicating that Cl 2 gas may have evaporated from the reaction medium during the formation of MnO 2 19 . Although the EDS analysis showed a minor ratio of the added materials, the SEM images showed a major effect on the morphology of the resulted fiber. The investigation of the crystal structure of the silk was performed using XRD as presented in Fig. 2(F) . The XRD patterns show that all the resulted silk has a mesophase behavior with a broad peak around 20.0°, which can be attributed to the β-sheet of silk II structure 21 – 25 . The mesophase structure of the silk is believed to facilitate the diffusion of ions to the internal parts of the silk fibers. Figure 2 Morphological and structural analysis of the silk: (A–E) FESEM images of the fabricated fibers (inset: cross section in the fiber) “pseudo-color is used for clarity” (A) S/B, (B) S/G, (C) S/MoS 2 , (D) S/TiO 2 , (E) S/Mn, and (F) the corresponding XRD patterns. Table 1 EDS analysis of the spun silk. Material C (atom%) N (atom%) O (atom%) Mo (atom%) S (atom%) Ti (Atom%) Mn (atom%) K (atom%) Cl (atom%) S/B 81.94 10.31 7.75 N/A N/A N/A N/A N/A N/A S/G 82.24 9.84 7.92 N/A N/A N/A N/A N/A N/A S/MoS 2 81.9 10.13 7.89 0.03 0.05 N/A N/A N/A N/A S/TiO 2 85.96 6.86 7.15 N/A N/A 0.03 N/A N/A N/A S/Mn 81.36 9.55 9.06 N/A N/A N/A 0.02 0.01 N/A As the Raman spectroscopy has been used as a good tool to investigate the deformation of polymers backbone structure 26 , the Raman spectra of the fabricated silk were recorded as shown in Fig. 3(A) . All fibers showed the same peak position with different intensities, indicating more or less a similar internal structure. The Raman active peaks of the studied fibers are in the range between 800 to 1800 cm −1 , in a good agreement with literature 4 , 26 . The observed Raman peaks of the B. Mori silk appeared at 1085, 1232 and 1669 cm −1 as indicated by red arrows in Fig. 3(A) . The FTIR spectra in Fig. 3(B) showed the typical peaks at 1623, 1515 and 1230 cm −1 characteristic of the silk fibers but with different intensities for different samples 6 , 8 , 9 . The peak at 1623 cm −1 indicated the presence of amide I structure, which can be ascribed to the vibration of the C=O bond due to the co-formation of the α-helix and random coiled structures. The peak at 1515 cm −1 indicated the presence of amide II structure, which can be related to the deformation of the N-H bond in the β sheet structure. The Peak at 1623 cm −1 indicated the presence of amide III structure and the peak is due to the vibration of the O-C-O bonds and the N-H bond. The positions of the peaks did not change with the chemical additives, which confirm that the chemical additives did not change the original backbone of the silk fibroins and hence the mechanical properties. CasaXPS software 27 was used to deconvolute the peaks of the FTIR, which indicated that the percentage of both α-helix and β sheet structure are almost equal in all samples and the presence of the β-sheet structure was found to be more pronounced in the S/TiO 2 , the blank silk, and the S/G samples more than in the S/Mn, and the S/MoS 2 samples, the deconvoluted data are presented in Table S1 . Thermogravimetric analysis was performed to indicate the thermal stability of the resulted silk fibers. Figure 3(C) shows that all the silk fibers were stable up to 200 °C then the blank silk started to decompose at ~250 °C. The modified silk showed enhanced thermal stability. At 500 °C, the remaining weight of the silk was 28.18, 26.7, 21.67, 16.13 and 12.27% for S/TiO 2 , S/G, S/MoS 2 , S/Mn, and S/B, respectively. Figure 3 The characterized peaks of the spun silk fibers (A) Raman spectroscopy, (B) FT-IR, and (C) TG analysis for the silk fibers. Electrochemical performance of the natural silk To test the capacitive performance of the natural silk, the self-standing silk was tested once as a positive electrode and once as a negative electrode in a 3-electrode system with 6 M KOH as the electrolyte. Although 6 M KOH is a high concentration electrolyte, it is commonly used with the carbon-based materials in supercapacitor applications 28 – 33 . Usually, the carbon materials show a typical rectangular cyclic voltammogram (CV) reflecting the electrical double layer behavior (EDL) 34 – 36 . However, the CVs of the positive and negative silk electrodes in Fig. 4(A,B) did not show an EDL behavior indicating diffusion processes for the ions in the polymeric structure of the silk 34 . It is expected that the OH- ion from the KOH reacted with the organic polymer of the silk fibers resulting in a diffusion and pseudocapacitive behavior to the silk electrodes. The ions from the KOH can react with MoS 2 , TiO 2 , and MnO 2 to give MoSSOH 37 , TiOOK 38 and MnOOK 39 , respectively. The CVs of the positive silk electrodes at a scan rate of 10 mV/s (Fig. 4(A) ) show that the redox peaks are more visible in the S/TiO 2 while the other additives did not affect the shape of the CV of the S/B. This can be ascribed to the accumulation of TiO 2 on the surface of the silk fibers while other additives affected the morphological shape of the silk fibers and did not accumulate with high amount on the surface of the fibers. At a scan rate of 10 mV/s and at a positive potential window, the specific capacitance of the S/Mn showed the highest specific capacitance of 778.975 mF/g while the S/TiO 2 , S/MoS 2 , S/G and S/B showed 577.925, 419.767, 247.822, and 157.291 mF/g, respectively. This shows that all the additives dramatically increased the specific capacitance values of silk electrodes. The CVs of the negative silk electrodes at a scan rate of 10 mV/s (Fig. 4(B) ) show clearer redox peaks than the positive electrodes. The specific capacitance of the negative electrodes calculated at a scan rate of 10 mV/s was 1122.832, 263.047, 131.794, 112.141, and 109.403 mF/g for S/Mn, S/B, S/MoS 2 , S/G and S/TiO 2 , respectively. To make a deeper study with accurate weight of the active material, the strands of the silk fibers were coiled over a glassy carbon (GC) electrode and measured as a positive electrode. The calculated specific capacitance of silk fibers @ GC at 10 mV/s (Fig. 4(C) ) showed a capacitance of 610.911, 604.701, 569.047, 556.923, and 206.650 mF/g for S/MoS 2 , S/TiO 2 , S/Mn, S/G and S/B, respectively. The contribution of the GC current collector affected the shape of the CVs and shifted them to the EDL rectangular shape. Also, the GC affected the values of the specific capacitance and the order of the materials in their capacitance values. Therefore, the current collector affects greatly the overall performance of the material and we will focus herein on the self-standing fibers as they are more reliable for the study. As one of the most important metrics of supercapacitors is their ability to store and release charges, the time of the charge and discharge was also studied for the silk fibers. Figure 4(D,E) shows the galvanic charge/discharge (GCD) curves of the self-standing silk fibers at a current density of 0.1 A/g. The GCD curves show a pseudocapacitive behavior 34 . For the positive electrodes, the specific capacitance calculated from the GCD at 0.1 A/g showed the same trend as that calculated from the CVs at 10 mV/s. The specific capacitance values of the positive electrodes calculated at 0.1 A/g were 1222.2, 373.25, 177.2, 54.6, and 29.25 mF/g for S/Mn, S/TiO 2 , S/MoS 2 , S/G and S/B, respectively. However, for the negative electrodes, the specific capacitance values calculated at 0.1 A/g were 3114, 108.7, 53.6, 37.05, and 17.8 mF/g for S/Mn, S/TiO 2 , S/B, S/MoS 2 and S/G, respectively. The GCD curves of the silk @ GC positive electrodes at 0.1 A/g are presented in Fig. 4(F) . The specific capacitance of the positive silk @ GC calculated at 0.1 A/g were 88.3, 85.1, 81.05, 68.1, and 35 mF/g for S/TiO 2 , S/MoS 2 , S/Mn, S/G and S/B, respectively. As for the CV results of the silk @ GC, the trend is different, and the effect of the current collector is shifting the shape of the GCD curves to the ideal shape of the EDL capacitor materials. However, the specific capacitance values of the silk with additives are still much higher than this of the blank silk. The CV and GCD results showed that the blank silk (S/B) behaved better as a negative electrode than as a positive electrode and so did the addition of Mn ions (S/Mn) and usually MnO 2 acts as a better capacitive material when used as a negative electrode 19 . However, the S/G, S/MoS 2 and S/TiO 2 enhanced the performance of the silk as a positive electrode than as a negative electrode. Although the amounts of the additives were relatively low, their effect can be attributed to both the nature of the materials and their effect on the morphology of the silk fiber, which controls the diffusion of ions into the silk fibers. Figure 4 Electrochemical performance of the silk fibers: (A) CVs of the studied self-standing silk fiber at 10 mV/s in positive potential window (inset: legend of (A–F) ), (B) CVs of the studied self-standing silk fiber at 10 mV/s in negative potential window, (C) CVs of the studied silk @ GC at 10 mV/s in positive potential window, (D) GCDs of the studied self-standing fibers at 0.1 A/g in positive potential window (inset: enlarged figure), (E) GCDs of the studied self-standing fibers at 0.1 A/g in negative potential window (inset: enlarged figure), (F) GCDs of the studied silk @ GC at 0.1 A/g in negative potential window. The conductivity is one of the main factors that affects the overall performance of a supercapacitor electrode. Figure 5(A) shows the Nyquist plots of 10 mg of silk fibers coiled over the same area of a glassy carbon electrode. The resulted curves were fitted to the inset circuit in Fig. 5(A) , with R1 representing the electrolyte resistance and R2 representing the charge transfer resistance of the material. As the obtained circle is a depressed semicircle not a perfect semicircle, Q was used in the fitting instead of C and L is used to represent the inductance related to the electrical connections, Z’ and Z” represent the real part and the imaginary part of the impedance, respectively. This circuit showed a perfect match with all the Nyquist plots as presented in Fig. S1 . The R2 values of the silk fibers were 157.7, 115.7, 104.9, 92.54, and 39.72 Ω for S/B, S/G, S/TiO 2 , S/MoS 2 and S/Mn, respectively. Those R2 values show that the additives greatly enhanced the conductivity of the silk fibers and hence enhanced their specific capacitance. The supercapacitors should be able to work under different conditions of scan rates and current densities. The value of the specific capacitance of self-standing silk positive electrodes versus the scan rate is presented in Fig. 5(B) . Note that the specific capacitance values have the same trend except at 500 mV/s. At 500 mV/s, the specific capacitance values are 196.991, 117.22, 87.491, 76.976, and 55.531 mF/g for S/Mn, S/MoS 2 , S/TiO 2 , S/G, S/B, respectively. On the other hand, from the GCD calculations of the positive self-standing silk electrodes (Fig. 5(C) ), the trends differed over the high current density. It showed the values of 78.25, 26.5, 9.5, 8.75, and 5.5 mF/g at 0.5 A/g for S/Mn, S/MoS 2 , S/G, S/TiO 2 , and S/B, respectively. For the negative self-standing silk electrodes, the change of specific capacitance with scan rate is presented in Fig. 5(D) . The values of the S/Mn and S/B were always much higher than those of the S/G, Si/MoS 2 , and Si/TiO 2 . At a scan rate of 500 mV/s, the specific capacitance values of the negative self-standing electrodes were 211.009, 60.195, 36.27, 35.729, and 30.272 mF/g for S/Mn, S/B, S/G, S/TiO 2 , and S/MoS 2 , respectively. The trend of the specific capacitance at different current densities is presented in Fig. 5(E) and enlarged in Fig. S2 . At a current density of 0.5 A/g, the specific capacitance values of the negative self-standing electrodes were 90.5, 12, 7.75, 6.8, and 5.25 mF/g for S/Mn, S/MoS 2 , S/G, S/TiO 2 , and S/B, respectively. Although the S/TiO 2 , S/MoS 2 , and S/G specific capacitance values as negative electrode (from GCD) are higher than that of the S/B but it is lower than their positive electrode values (from GCD). Thus, it is believed that S/TiO 2 , S/MoS 2 , and S/G act better as positive electrodes than as negative electrodes. Despite the different trends over the different scan rates and current densities, the performance of all silk with additives was better as positive electrodes than the blank silk and the S/Mn was always better as a negative electrode. One of the performance metrics of the supercapacitor materials is their stability upon cycling. Figure 5(F) shows the retention percentage of the self-standing silk as positive and negative electrodes over 1000 cycles. The retention fluctuates at the first 200 cycles and reaches a relative stability after 600 cycles. The positive electrodes showed retention of 141.88, 90.59, 87.7, 66.63, and 61.3% for S/TiO 2 , S/MoS 2 , S/G, S/Mn, and S/B, respectively after 1000 cycles. The negative electrodes showed retention of 80.99, 67.6, 63.45, 46.06, and 42.13% for S/MoS 2 , S/TiO 2 , S/Mn, S/B, and S/G, respectively. From the retention results we conclude that the silk fiber has a better retention as a positive electrode in general and that the additives enhanced the retention and cyclability of the electrodes. the above 100% retention values are attributed to the further diffusion of ions into the material and enhancement of reaction over time 17 , 40 . Noteworthy to mention that the specific capacitance values in mF are acceptable for self-standing carbon-based materials with no high conductive current collectors 41 – 43 . Figure 5 Electrochemical stability of the silk fibers (A) Nyquist plots of the studied silk @ GC in the range 1 MHz to 100 mHz (inset: fitting circuit and fitting curve). (B) Change of specific capacitance with scan rate (10, 50, 100, and 500 mV/s) for the self-standing fiber in positive potential window (inset: legend for (A–E) ), (C) Change of specific capacitance with current density (0.1, 0.2, 0.4 1nd 0.5 A/g) for the self-standing fiber in positive potential window, (D) Change of specific capacitance with scan rate (10, 50, 100, and 500 mV/s) for the self-standing fiber in negative potential window, (E) Change of specific capacitance with current density (0.1, 0.2, 0.4 1nd 0.5 A/g) for the self-standing fiber in negative potential window, (F) Retention of the studied self-standing fiber in both positive and negative potential window."
} | 5,510 |
26388858 | PMC4560021 | pmc | 4,174 | {
"abstract": "The set-up of biorefineries for the valorization of lignocellulosic biomass will be core in the future to reach sustainability targets. In this area, biomass-degrading enzymes are attracting significant research interest for their potential in the production of chemicals and biofuels from renewable feedstock. Glutathione-dependent β-etherases are emerging enzymes for the biocatalytic depolymerization of lignin, a heterogeneous aromatic polymer abundant in nature. They selectively catalyze the reductive cleavage of β- O -4 aryl-ether bonds which account for 45–60% of linkages present in lignin. Hence, application of β-etherases in lignin depolymerization would enable a specific lignin breakdown, selectively yielding (valuable) low-molecular-mass aromatics. Albeit β-etherases have been biochemically known for decades, only very recently novel β-etherases have been identified and thoroughly characterized for lignin valorization, expanding the enzyme toolbox for efficient β- O -4 aryl-ether bond cleavage. Given their emerging importance and potential, this mini-review discusses recent developments in the field of β-etherase biocatalysis covering all aspects from enzyme identification to biocatalytic applications with real lignin samples."
} | 313 |
25848808 | null | s2 | 4,176 | {
"abstract": "Direct solar-powered production of value-added chemicals from CO2 and H2O, a process that mimics natural photosynthesis, is of fundamental and practical interest. In natural photosynthesis, CO2 is first reduced to common biochemical building blocks using solar energy, which are subsequently used for the synthesis of the complex mixture of molecular products that form biomass. Here we report an artificial photosynthetic scheme that functions via a similar two-step process by developing a biocompatible light-capturing nanowire array that enables a direct interface with microbial systems. As a proof of principle, we demonstrate that a hybrid semiconductor nanowire-bacteria system can reduce CO2 at neutral pH to a wide array of chemical targets, such as fuels, polymers, and complex pharmaceutical precursors, using only solar energy input. The high-surface-area silicon nanowire array harvests light energy to provide reducing equivalents to the anaerobic bacterium, Sporomusa ovata, for the photoelectrochemical production of acetic acid under aerobic conditions (21% O2) with low overpotential (η < 200 mV), high Faradaic efficiency (up to 90%), and long-term stability (up to 200 h). The resulting acetate (∼6 g/L) can be activated to acetyl coenzyme A (acetyl-CoA) by genetically engineered Escherichia coli and used as a building block for a variety of value-added chemicals, such as n-butanol, polyhydroxybutyrate (PHB) polymer, and three different isoprenoid natural products. As such, interfacing biocompatible solid-state nanodevices with living systems provides a starting point for developing a programmable system of chemical synthesis entirely powered by sunlight."
} | 421 |
35407295 | PMC9000898 | pmc | 4,177 | {
"abstract": "In this study, we fed the larval of Bombyx mori silkworms with nanodroplets of liquid metal (LM) coated with microgels of marine polysaccharides to obtain stretchable silk. Alginate-coated liquid metal nanodroplets (LM@NaAlg) were prepared with significant chemical stability and biocompatibility. This study demonstrates how the fed LM@NaAlg acts on the as-spun silk fiber. We also conducted a series of characterizations and steered molecular dynamics simulations, which showed that the LM@NaAlg additions impede the conformation transition of silk fibroins from the random coil and α-helix to the β-sheet by the formation of hydrogen bonds between LM@NaAlg and the silk fibroins, thus enhancing the elongation at the breakpoints in addition to the tensile properties. The intrinsically highly stretchable silk showed outstanding mechanical properties compared with regular silk due to its 814 MPa breaking strength and a breaking elongation of up to 70%—the highest reported performance so far. We expect that the proposed method can expand the fabrication of multi-functional silks.",
"conclusion": "4. Conclusions In this work, we demonstrated that intrinsically reinforced silks can be simply produced by feeding silkworms with LM@NaAlg. Compared with control silk, the breaking strength and breaking elongation of the treated LM@NaAlg silk reached up to 814 MPa and 70%, respectively, which is the highest reported performance so far. This might be attributed to the formation of hydrogen bonds between LM@NaAlg and silk fibroin, leading to increased random coil/α-helix structures, higher orientation, and fewer β-sheet structures. In addition, we also examined the incorporation of LM@NaAlg, and it appeared to have a negative influence on the crystalline structure and conformation of silk fibers. Overall, this work provided a straightforward and effective way of improving the mechanical performance of silk. It might also shed light on the in vivo study of the liquid metal transportation mechanism and facilitate the large-scale production of reinforced silks. It is worth noting that several questions exist in our current investigation. For example, how to quantify and improve the efficiency of the LM@NaAlg uptake by the silkworms, how LM@NaAlg affects the silk structures in biological processes, and how LM@NaAlg is transported from mid-gut, hemolymph, and the silk gland into the silk. We also expect that researchers may be inspired to perform bio-interactions between functionalized nanomaterials and silkworms on the molecular, cellular, tissue, and even organ levels.",
"introduction": "1. Introduction Silk has been used as a natural filament fiber for hundreds of years. It has been widely used in the biomedical field, textile industry, and even as an engineering material due to its favorable biocompatibility, controllable biodegradability, lustrous appearance, and excellent mechanical properties [ 1 , 2 , 3 , 4 ]. Various functional components, such as fluorescent proteins [ 5 ], rare-earth upconverting phosphors [ 6 ], antimicrobial agents [ 7 , 8 ], metal ions [ 9 ], metal and semiconductor nanodroplets [ 10 , 11 ], and graphene quantum dots [ 12 , 13 , 14 ], have been used to intrinsically produce functionalized silks. Extrinsic and intrinsic functionalization approaches have been used to improve the performance of silk. Traditional extrinsic functionalization methods add modifiers to the surface of silk [ 15 , 16 , 17 , 18 ]—which inevitably requires the use of toxic chemicals—or re-spin the structure of regenerated silk using additives [ 19 , 20 ]. Many researchers have been devoted to finding an alternative way to incorporate external substances into silkworm silk to enhance its mechanical properties. There are mainly three methods used for improving the properties of silk fibers: gene overexpression [ 21 , 22 ], feeding [ 23 , 24 , 25 ], and injecting [ 26 , 27 ]. Lizuka et al. reported that silkworms used for the mass production of three colors (green, red, and orange) of fluorescent silk can be generated using a vector originating from the fibroin H chain gene [ 28 ]. Although significant performances were realized through genetic alterations, the synthesis procedures are complicated and very costly. In comparison, the in vivo uptake, feeding, and injecting approaches are much easier and cheaper. Ma et al. successfully increased the toughness modulus of silks by the intravascular injection of albumin bovine (BSA)-stabilized gold nanoclusters [ 29 ]. Compared with injections, which are lethal and not suitable for large-scale production, feeding specific diets to silkworms is among the most common approaches to functionalizing silk fibers due to its convenient and green properties. Several groups have acquired enhanced stretchable silks by feeding additives, including amino acids [ 30 ], dyes [ 31 , 32 ], and nanomaterials [ 12 , 23 , 25 ], to silkworms. For example, Wang et al. revealed that high-strength silks can be directly obtained by feeding silkworms with graphene nanosheets [ 25 ]. Wu et al. characterized the impact of the mechanical properties of the resulting silk fibers from silkworms fed with different nanoparticles (Cu, Fe, and TiO 2 ), and the obtained Cu-containing silk fibers exhibited a good tensile strength of 360 MPa and reached a strain of 38% [ 25 ]. Although the reported nanomaterial feeding methods increased the silk toughness, the rigid intrinsic property of the used nanomaterials restricted the stretchability extent. Thus, further improving the stretchability performance of silk remains an essential research area. Liquid metal possesses both “liquid” and “metallic” properties [ 33 ], and it is a promising material for soft bioelectronics due to its excellent conductivity, stretchability, super compliance, low cost, and environmental processing technology. Compared with traditional stiff materials, the emerging gallium-based liquid metals with the properties of low Young’s moduli (1–10 Pa), which are infinitely deformable in principle, have drawn a great deal of attention as ideal candidates for fabricating highly stretchable devices [ 34 , 35 ]. However, to our best knowledge, feeding silkworms with liquid metal has not been explored. In this study, directly from the second day of their fifth instar, we fed Bombyx mori silkworms with diets containing nanodroplets of alginate-coated liquid metal (LM@NaAlg), which significantly improved the tensile property of silk more efficiently than that of the reported rigid materials, to obtain intrinsically reinforced silkworm silk fibers. The alloys of Ga, In, and Sn were used as liquid metals in this work. The conducted cytotoxic experiment indicated that the LM@NaAlg nanodroplets had no obvious negative effects on the growth status of silkworms. Also, the silk properties were characterized by analyzing their thermal stability, and the silk structures were examined using a scanning electron microscope (SEM). We studied the dissolved silk fibers using Fourier transform infrared (FTIR) spectra and X-ray diffraction (XRD), and it was confirmed that the incorporation of parts of LM@NaAlg into the as-obtained silk fibers was successful. The evidence for the mesophase in silkworm silks was demonstrated by synchrotron radiation small-angle X-ray scattering (SR-SAXS). Two-dimensional wide-angle X-ray diffraction (2D-WAXD) and Raman spectra were used to study the conformational changes in the obtained silk fibers. Furthermore, steered molecular dynamic simulations were performed to confirm the experimental results. Finally, the conducted strain–stress of the obtained silk fibers was measured.",
"discussion": "3. Results and Discussion 3.1. The Preparation of LM@NaAlg Nanodroplets and Effect on Silk Fibers As shown in Figure 1 a, aiming at hindering the rapid oxidation of the liquid metal when exposed to water and oxygen, at the same time, improving the biocompatibility, we first produced a stable aqueous ink of liquid metal by coating liquid metal nanodroplets with sodium alginate. It was reported that the carboxylic groups within the alginate G segments could chelate with multivalent cations, producing an “egg-box” structure and gelling alginate solutions [ 37 ]. The sodium alginate-protected liquid metal facilitated the downsizing process of liquid metal due to the coordination of their carboxyl groups with Ga 3+ . In this case, it formed microgel shells around liquid metal droplets by chelating Ga 3+ into structural “egg-box” crosslinkers. After 60 min of sonication, a stable opaque slurry was obtained, and the LM@NaAlg nanodroplets were obtained after washing and size-grading with a diameter of 40–250 nm and an average value of 116 nm ( Figure 1 b,c). The thickness of the alginate layer coated on the surface of the liquid metal droplets was around 20 nm, which was measured from the TEM image of the LM@NaAlg ( Figure 1 b), and the uniformity of the sodium alginate layer was excellent, though the sizes of LM@NaAlg nanodroplets were not uniform. The LM@NaAlg nanodroplets were stable without coalescing and oxidizing for a long period of time of >7 d in the air due to their mechanical robustness and the decreasing oxygen permeability of the microgel shells. The stability of the LM@NaAlg nanodroplets was shown in Figure S1 , and the TEM image measured after six days’ preservation continued to present excellent morphology with the undestroyed shell of sodium alginate. However, precipitation began to appear on day 8, and it was more obvious on day 10. Next, MTS assays were conducted to evaluate the cytotoxicity of the LM@NaAlg to HeLa cells and 4T1 cells. As shown in Figure 2 , the Hela cell viability was estimated to be greater than 95% with 100 μg mL −1 of LM@NaAlg for 24 and 48 h, indicating the obtained remarkable biocompatibility. After being cultivated with different concentrations of LM@NaAlg nanodroplets for 24 h and 48 h, 4T1 cells were in good shape, but a negligible decrease could be observed when the 4T1 cells were incubated for 48 h, potentially attributed to the excessive consumption of the culture medium. Overall, the significant biocompatibility of the LM@NaAlg paved the way to produce stretchable silk as the diet of silkworms. 3.2. Effect of LM@NaAlg in Feeding Diet on Growth and Silk of Silkworms We separated 80 silkworms into four groups, each containing 20 silkworms with similar body weights. Three experimental groups were fed with mulberry leaves sprayed with an LM@NaAlg solution concentration of 1, 2, and 3 wt%, named LM@NaAlg-1, LM@NaAlg-2, LM@NaAlg-3, respectively. No obvious differences between the silkworms fed with different diets were observed until cocoons were produced, demonstrating that the diets containing the used LM@NaAlg nanodroplets in this work were safe for raising silkworms ( Figure 3 a–d). Also, the photographs of the as-obtained cocoons shown in vignettes exhibited similar colors and uniform sizes. The average silkworm weight mass was recorded 7 days after feeding the additives of the LM@NaAlg nanodroplets ( Figure S2 ), and all the silkworms had similar weights over the 7 days duration of the fifth instar with no obvious differences between those fed with the LM@NaAlg nanodroplets diet and the control groups. The silkworm larvae survival rate for each group demonstrated that the LM@NaAlg had a negligible effect with a mortality rate of 0.05% for the LM@NaAlg-2 and LM@NaAlg-3 groups ( Table S1 ). This demonstrated that the modified diets in our work were safe for silkworms. In addition, the cocooning rate, cocoon shell weight, and silk diameter were also studied ( Figures S3–S5 ) to further investigate the security of the used LM@NaAlg nanodroplets. The cocooning rate was slightly different due to the different uptake of the various LM@NaAlg concentrations. The biggest silk diameter (2 wt% group) may be attributed to the high breaking elongation data, and the SEM image of silk fiber cross-sectional shapes is shown in Figure S6 . This can be considered the optimal concentration for the LM@NaAlg nanodroplets uptake in our work. All the silk cocoons were degummed to remove the sericin coating on the silk fibers, so they could be used in the following characterization. The diameter and morphology of the degummed silks were characterized using an SEM. As shown in Figure 2 e–h, we found that the silks generated from LM@Alg-1, LM@Alg-2, and LM@Alg-3 exhibited similar morphology compared with the control group, indicating that the incorporation of LM@NaAlg nanodroplets did not have an apparent influence on the silk morphology. The results of the energy-dispersive spectra (EDS) mapping of the modified silk displayed the uniform distribution of the content and the distribution of the gallium (Ga), indium (In), and stannum (Sn) elements in the silk fiber ( Figure 2 i–l), indicating that LM@NaAlg perfectly combined with the silks. The content distributions of the different elements in the modified silk fibers were further investigated. Ga (1.31 wt%), In (0.46 wt%), and Sn (0.42 wt%) were obviously detected, while C (60.19 wt%) and O (34.91 wt%) were dominant, indicating the successful combination of liquid metal and silk fibers ( Figure S7 and Table S2 ). 3.3. Thermal Degradation of Degummed Silks The thermal stability of the modified silk fibers was measured using thermogravimetric analysis (TGA) under a nitrogen atmosphere at a scanning speed of 15 °C min −1 . The results of the TGA and differential thermos gravimetric (DTG) curves showed the mass change in the modified silk fibers during the heating process from 30 °C to 800 °C ( Figure S7 ). The intermolecular-bound residual water in the silk fibers was removed as the temperature increased to 150 °C, and the silk fibers exhibited similar thermal degradation curves with a rapid mass decrement from around 295 °C. Specific significant weight loss temperatures are shown in Figure S8 . The DTG curves were the first derivatives of the corresponding TAG curves. When the silk was heated, the molecules in the amorphous region of the fiber were first moved. As the temperature increased, the molecular chain in the crystallization zone gradually moved, and the macromolecular chain was cleaved [ 34 ]. For the control group, the starting and highest decomposition temperatures of the decomposition were 291.58 °C and 329.81 °C, respectively. These decompositions occurred at higher corresponding temperatures for the LM@NaALg-1 group at 294.71 °C and 329.87 °C, respectively, for the LM@NaALg-2 group at 297.66 °C and 322.65 °C, respectively, and for the LM@NaALg-3 group at 295.25 °C and 348.4 °C, respectively, owing to the liquid metal nanodroplets in the silk fibers. The TGA and DTG results thus prove that the addition of liquid metal nanodroplets in the silk fibers using modified LM@NaAlg nanodroplet diets can enhance the thermal stability and slow the thermal degradation of silkworm silk. 3.4. Secondary Structure Characterizations of Degummed Silks Fourier-transform infrared spectroscopy (FTIR) is one of the most powerful methods for characterizing the second structure and super inter-interactions of silk fibers ( Figure 4 a) [ 11 , 34 ]. The signal at 1227 cm −1 was assigned to the β-sheets and random coils or/and α-helixes [ 38 ]. Also, the signal at 1617 cm −1 was considered to be attributed to the β sheet conformation, and the absorb peak at 1513 cm −1 was ascribed to the β-sheet structure due to the N-H deformation [ 38 ]. The identical peak position of the FTIR spectra confirmed that the LM@NaAlg nanodroplets did not have covalent interactions between the basic structure of the silk fibers and the LM@NaAlg nanodroplets. Fourier self-deconvolution (FSD) was conducted for the corresponding content of the amide I regions to quantify the β-sheet, random coils/α-helix, and β-turn contents to demonstrate the nano–bio interactions in the silk fibers ( Figure 4 b–f). The contents of the random coil and the helix of LM@NaAlg-1 were 0.41 and 0.38 in the LM@NaAlg-3 group, respectively, while the contents of the β-sheet were 0.35 and 0.4, respectively. Both were higher than the control group (0.33). The LM@NaAlg-modified silks contained a greater number of chains in the random coil/α-helix and β-turn conformations compared with the control silk. The β-sheet content of the control group was approximately 33.7%, which was better than that of the LM@NaAlg-2 group (32.1%). This may be ascribed to the abundant carboxyl and hydroxyl on the surface of LM@NaAlg nanodroplets, which were in favor of forming hydrogen bonds with the amino groups of silk fibers and slightly hindered the conformation transition of the silk protein from random coil/β-turn to β-sheet, corresponding to the better strength and stiffness. The proposed reinforcing schematic illustration shows the interactions between the LM@NaAlg nanodroplets and silk fibers ( Figure 4 g–h). During stretching, the random coil/α-helix conformational chains were the first to deform in the amorphous phase because they were easily movable. At the same time, the nanometer size scale, intensive hydrogen bond interactions, and spherical morphology caused the LM@NaAlg nanodroplets to move with the protein chains, providing more space for the chains to move, while about 28% and 15% of hydrogen bonds between protein–protein in the control and the LM@NaAlg-modified group were destroyed during the first stretching process. After 12 s, the proportion of it stood at 50% and 32%, respectively. This collaborative mobility promoted a larger elongation at the break to the modified silk fibers. Furthermore, the higher orientation and increased content of mesophase further enhanced the mechanical properties of the LM@NaAlg-modified silk fibers. 3.5. Steered Molecular Dynamics of LM@NaAlg-Modified Silk Fibers Steered molecular dynamic (SMD) simulations are performed to further investigate the mechanical properties of the LM@NaAlg-modified silk fibers. The left end of the silk fiber was fixed and stretched the middle of the rest portion, shown in Figure 5 a. In the process of silk stretching, the system was more easily broken up without LM@NaAlg, while it was relatively stable with LM@NaAlg; after 4s, the difference could be observed. The secondary structure of the silk fiber changed during the stretching process, as shown in Figure 5 b,c, which further highlights the LM@NaAlg’s contribution to the stretchable silk. Figure 5 d shows the change of tension with time. When LM@NaAlg exists, the system generates stress later and becomes more stable, which can essentially be attributed to the H-bond of the silk fiber shown in Figure 5 e. The H-bond number of LM@NaAlg-contained silk was gradually increased, rose slightly, and then leveled off, but the control group was declined until the structure was completely destroyed. The statistics in Figure S9 can also prove this. The SMD simulations directly performed the stretching silk combined with LM@NaAlg. 3.6. Crystalline Structure Characterization of Silk Fibers It is essential to discuss the role of the interface phase, or mesophase, which acts as a modulus intermediate between the amorphous and crystalline phases and is of great significance in influencing the mechanical properties of silk fibers. To further characterize the crystalline structure of the silk fibers, 2D-WAXD and XRD were used, and the resultant patterns are shown in Figure 6 . As seen from Figure 6 a and Figure S10 , the 1D and 2D WAXD patterns do not show obvious differences but differ in intensity. This is true for the highly stretchable silk, and mainly because of this the modification of LM@NaAlg impedes the conformation transition of silk fibroins from the random coil and α-helix to the β-sheet by the formation of hydrogen bonds between LM@NaAlg and the silk fibroins, thus enhancing the elongation at the breakpoints, in addition to the tensile properties. Hance increased the intensity of WAXD. Furthermore, the evidence for the mesophase in silks was also implied by SR-SAXD. The 2D SR-SAXD patterns were shown in Figure S11 . Figure 6 b illustrates the 1D SR-SAXD spectra of the silks. The inset pattern was the profiles of q2I(q)−q-2 based on the modified Porod law, which was used to calculate the interface factor. The interface thickness was approximately calculated and is listed in Table S3 [ 39 ]. The inset of Figure 6 b suggests that the interface factors of the modified silks are larger than that of the control group. Since the interface factor was in direct proportion to the thickness of the interface, it is rational to consider that LM@NaAlg-modified silks have a thicker interface than control silk. The thickness of the interface increased to the highest figure with LM@NaAlg-2. However, when the concentration increased to LM@NaAlg-3 or declined to LM@NaAlg-1, the thickness of the interface decreased a little, which might be attributed to the poorer combination between silk fibroin and LM@NaAlg. All the XRD patterns showed two typical diffraction peaks at around 20.5° ( Figure 6 c), which were attributed to the characteristic peaks of the β-sheet crystalline structure [ 40 , 41 ]. There was no noted difference among these patterns, regardless of the LM@NaAlg nanodroplet concentrations in each group. This phenomenon was consistent with the results of FTIR, indicating that the basic structures of silk fibers were barely changed by feeding LM@NaAlg nanodroplets. To confirm the conformational changes in the obtained silks upon the intake of LM@NaAlg in the silk cocoons, Raman spectroscopy was performed ( Figure 6 d). For the silks from either the LM@NaAlg or control groups, the most prominent Raman-active bands were observed at the same positions. The peak at 1,084 cm −1 represents the random coil conformation, and the peak at 1231 cm −1 could be assigned to the predominantly β-sheet conformation. The observed peak at 1666 cm −1 corresponds to the β-sheet/β-turn conformation, and the peaks at 2936 cm −1 and 3986 cm −1 correspond to the CH 3 asymmetric stretch and N-H stretch, respectively [ 41 ]. In general, a similar intensity ratio and peak position were obtained, indicating that the conformation of the silks barely changed with the intake of the LM@NaAlg nanodroplets. 3.7. Stretchable Mechanical Properties of LM@NaAlg-Modified Silks The mechanical properties, including the breaking strength and elongation at the breakpoints of the silks, were closely associated with their secondary structures [ 42 , 43 ]. The typical stress–strain curves of the silk fibers shown in Figure 7 demonstrate the changes in the mechanical properties of the modified silks. It was observed that the LM@NaAlg-modified silks had significantly improved tensile properties compared with the control silks. The LM@NaAlg-1 silks exhibited a breaking strength of 420.43 MPa and elongation at a break of 36.58%. Also, the LM@NaAlg-3 silks possessed a breaking strength of 665.13 MPa and elongation at a break of 33.2%, considerably exceeding the values of control silks (196.14 MPa, 26.3%). When the silkworms were fed with an LM@NaAlg solution concentration of 2 wt%, a breaking elongation of up to 70% and a breaking strength of 814 MPa were realized, which are the highest values obtained so far ( Table 1 ). As shown in Figure 7 b,c, the average strain and stress measurements demonstrate the same trend that was accepted from the three-time measurement of the silk fiber in different groups. The remarkable improvement in the mechanical properties may be attributed to the excellent ductility of liquid metal and the high biocompatibility of LM@NaAlg nanodroplets. However, the high content of LM@NaAlg may aggregate and act as a defect, causing a low breaking strength or elongation at break."
} | 5,956 |
37217549 | PMC10203214 | pmc | 4,178 | {
"abstract": "Efficient valorization of lignin, a sustainable source of functionalized aromatic products, would reduce dependence on fossil-derived feedstocks. Oxidative depolymerization is frequently applied to lignin to generate phenolic monomers. However, due to the instability of phenolic intermediates, repolymerization and dearylation reactions lead to low selectivity and product yields. Here, a highly efficient strategy to extract the aromatic monomers from lignin affording functionalized diaryl ethers using oxidative cross-coupling reactions is described, which overcomes the limitations of oxidative methods and affords high-value specialty chemicals. Reaction of phenylboronic acids with lignin converts the reactive phenolic intermediates into stable diaryl ether products in near-theoretical maximum yields (92% for beech lignin and 95% for poplar lignin based on the content of β−O−4 linkages). This strategy suppresses side reactions typically encountered in oxidative depolymerization reactions of lignin and provides a new approach for the direct transformation of lignin into valuable functionalized diaryl ethers, including key intermediates in pharmaceutical and natural product synthesis.",
"introduction": "Introduction Among the plentiful biomass, lignin is the only sustainable source of aromatic compounds 1 – 6 and is available in abundant quantities as a waste product from the pulp and paper and bioethanol industries 6 . Nonetheless, lignin is under-exploited as a renewable chemical feedstock due to the limited number of efficient and selective downstream processing strategies available. Various methods have been extensively studied for the conversion of lignin into aromatic products that can broadly be classified as catalytic oxidative degradation, catalytic reductive degradation and acid/base-catalyzed degradation 2 – 4 , 6 . Catalytic oxidative degradation has a number of advantages compared to other catalytic fractionation methods including hydrogenolysis (a reductive process), and acid/base-catalyzed degradation 4 , 7 , 8 . Catalytic oxidative degradation advantageously take place under mild and environmentally benign conditions, which contrasts with hydrogenolysis that uses noble metal catalysts, high reaction temperatures and pressures, or the use of corrosive acid/base-catalyzed degradation reagents. In addition, catalytic oxidative degradation has the potential to retain key functionality in the products that could be relevant in subsequent synthetic steps 9 – 11 . Many homogeneous and heterogeneous catalytic oxidative methods that cleave the C–C bonds of the alkyl side chains to depolymerize lignin have been reported, but typically they are limited by poor selectivity and consequently low product yields (Fig. 1 ) 12 – 23 . The catalysts reported for the oxidative fragmentation of lignin are summarized in Supplementary Table S1 .The critical issue that must be solved to overcome the aforementioned limitations is that the phenolic hydroxy group is unstable under oxidative conditions 24 , leading to side reactions including repolymerization and ring opening reactions, which generates complex polymers, oligomers, and non-aromatic side products 25 – 28 . Hence, aromatic products are isolated in low yields in certain direct oxidative degradation reactions. Fig. 1 Current catalytic oxidative degradation approach and the approach disclosed herein. COR 1 : −CHO or −COOH. R 2 : −H, −OMe, −CHO or −COOH. Diaryl ethers are typically prepared from cross-coupling reactions between petrochemical-derived substrates, specifically, phenols with excess electrophiles, i.e. aryl halides, or nucleophiles, i.e. boronic acids 29 . Inspired by Cu-catalyzed oxidative cross-coupling reactions between of nucleophiles (phenols, anilines, etc.) with organoboronic acids to afford poly-functionalized diaryl ethers and diaryl amines 30 – 34 , we decided to explore the utility of organoboronic acids to extract the reactive phenolic intermediates generated during the oxidative degradation of lignin (Fig. 1 ). We discovered that this protocol prevents common side reactions initialed by phenolic intermediates allowing functionalized diaryl ethers to be obtained in near-theoretical maximum yields. Using lignin as a starting material to synthesize functionalized diaryl ether is advantageous as lignin is an abundant, inexpensive and renewable material 1 – 6 . The direct conversion of lignin to diaryl ethers in a single step process requires fewer reagents and solvents than a two-step process in which phenols are initially generated from lignin and then further transformed 5 , 10 , 23 .",
"discussion": "Results and discussion Initially, organosolv beech lignin and 4-chlorophenylboronic acid ( 1a ) were reacted with O 2 in the presence of various Cu salts in a weakly alkaline solution that mimic the conditions typical of coupling 30 – 34 and C–C bond cleavage reactions 12 , 13 , 15 , 18 (Fig. 2a ). After the oxidative degradation/coupling step, a methylation step was performed to transforms any carboxylic acid groups into methyl ester groups, to facilitate gas chromatography (GC) analysis. From the Cu salts screened, copper(II) triflate (Cu(OTf) 2 ) was found to be the most effective catalyst together with bathophenanthroline (L1 in Fig. 2b ) co-catalyst, affording the diaryl ether products ( 1-4b ) in 92% yield (Fig. 2a , Entry 5). Note that other ligands were evaluated (Fig. 2a, b ), but none were as effective as L1. Since the triflate anions in Cu(OTf) 2 are weakly coordinating and readily displaced 35 , 36 . Cu(OTf) 2 is expected to react with L1, a bidentate N-donor ligand, to form the active catalyst in situ. L1 is an electron-rich ligand that is expected to increase the electron density on the Cu center, which facilitates oxidation Cu(II) to Cu(III), a key step in the reaction (the more electron rich the Cu center the easier it is to oxidize) 31 , 37 . A CuL1 complex is formed in situ (evidenced by mass spectrometry, Supplementary Fig. 1 ), which serves as the actual catalyst, and is sufficiently stable to be isolated after reaction, recycled and reused with only a minor loss in activity (Supplementary Fig. 1 ). The GC spectrum showing the product distribution is given in Fig. 2c and Supplementary Fig. 2 . As a representative hardwood 2 , the aromatic rings of beech wood consist of 79.5% syringyl (S), and 20.5% guaiacyl (G) units (Supplementary Fig. 3 ). The main diaryl ether products are the syringyl type methyl ester ( 1b , 68%) and the guaiacyl type methyl ester ( 3b , 15%), along with syringyl- and guaiacyl-type aldehyde products ( 2b , 8% and 4b , 1%, respectively), demonstrating that the aromatic rings of lignin may be extracted successfully as functionalized aromatic diaryl ether products using this method. Detail optimization of the reaction parameters is summarized in Supplementary Tables 2 – 11 . Fig. 2 Reaction of beech lignin with 4-chlorophenylboronic acid ( 1a ). a Reaction and optimization of the catalyst and co-catalyst, further details of the optimization of the reaction parameters are provided in the SI. Reaction conditions: (1) beech lignin (40 mg), 4-chlorophenylboronic acid (1.5 equiv.), Cu salt (30 mol%), co-catalyst (30 mol%), K 2 CO 3 (4 equiv.), biphenyl (0.02 mmol, internal standard), DMSO (2 mL), O 2 (3 atm), 140 °C, 6 h. (2) K 2 CO 3 (3 equiv.), MeI (10 equiv.), 25 °C, 12 h. The methylation step is required so the products can be analyzed by GC-MS. Yield of diaryl esters is based on the content of β–O–4 ether linkages in lignin. b Structures of co-catalysts. c GC spectrum of the reaction mixture (Entry 5, Fig. 2a) showing the presence of four products and their relative abundance. d Short-range HSQC spectra before and after reaction. Assignment of contours is provided by the numbering in the structures on the right. Overlying green contours in the HSQC spectra correspond to 1b . The evolution of the reaction was monitored by short-range 13 C- 1 H correlation (HSQC) NMR spectroscopy (Fig. 2d shows the spectra before and after reaction). The beech lignin structure consists of syringyl and guaiacyl aromatic rings, together with their main alkyl side chains (β–O–4 (A) and β–β (B) linkages) 2 , clearly identified in the HSQC spectrum before reaction. After reaction, signals corresponding to the alkyl side chains and electron-rich aromatic rings are no longer present, indicative of complete degradation of the lignin structure. The HSQC spectrum of the reaction mixture is consistent with that of the expected major product 1b (Fig. 2d , green contour) and other products, based on comparisons with full HSQC spectra of 1b – 4b (Supplementary Figs. 7 – 10 ). The remaining HSQC signals may be attributed to unreacted linkages of lignin and side products derived from the excess boronic acid used (Supplementary Fig. 2 ). In the absence of 1a , no aromatic monomer products were detected under the optimized reaction conditions, confirming that the boronic acid captures the reactive phenolic intermediates (Fig. 3 ). The composition and structure of lignin varies with the type of wood used (Supplementary Figs. 3 – 5 ), hence several types were evaluated (Fig. 3 ). Lignin from poplar, a hardwood, is primarily composed of S aromatic units. Poplar lignin was evaluated under the optimized reaction conditions, affording diaryl ethers in 95% yield, with high selectivity to the syringyl-type product 1b , which was obtained in 71% yield (Fig. 3 ). Pine lignin, containing G and coumaryl (H) aromatic units without S units, affords the G-type diaryl ether 3b as main product in 71% yield (Fig. 3 ). Raw beech wood sawdust was tested using the standard reaction conditions with diaryl ether products obtained in only 10% yield (Supplementary Fig. 11 ). Fig. 3 Aromatic ethers generated from lignin using different types of wood. Other side products corresponds to polymers, oligomers, and non-aromatic side products 25 – 28 . Standard reaction conditions: (1) Lignin (40 mg), 4-chlorophenylboronic acid (1.5 equiv.), Cu(OTf) 2 (30 mol%), L1 (30 mol%), K 2 CO 3 (4 equiv.), biphenyl (0.02 mmol, internal standard), DMSO (2 mL), O 2 (3 atm), 140 °C, 6 h. (2) K 2 CO 3 (3 equiv.), MeI (10 equiv.), 25 °C, 12 h. The scope of the boronic acid and related coupling reagents was also evaluated using beech wood lignin under the optimized reaction conditions (Fig. 4 ). Specifically, phenylboronic acid ( 2a ) affords the S type diaryl ether ( 6b ) in 66% yield. Phenylboronic acids functionalized with electron-withdrawing halogen ( 1a, \n 3 - 4a ) or trifluoromethyl ( 5 - 6a ) substituents are tolerated and afford the desired diaryl ether products in 64–71% yield. Nitro ( 7a ), ester ( 8a ), and methoxy ( 9a ) substituents at the para -position result in slightly lower yields (49–56%). Biphenyl boronic acid ( 10a ) was transformed in 61% yield. Several organic borate esters ( 11a – 13a ) were also tested and are less effective in the aromatic extraction reaction (yields ranging from 23 to 55%), and the phenyltrifluoroborate salt ( 14a ) and alkyl boronic acid ( 15a, \n 16a ) do not function as coupling reagents. In general, phenylboronic acids and borate esters could be employed as effective extraction reagents for the transformation of lignin into functionalized diaryl ether products. Diaryl ethers with ortho- methoxy and/or para- carboxy groups are commonly encountered as intermediates in the preparation of pharmaceutical and natural products, such as chrysophaentins 38 , 39 , himalain A 40 and certain biological inhibitors 41 , 42 . Fig. 4 Substrate scope of the boronic acid and related coupling reagents. Reaction conditions: (1) Beech lignin (40 mg), boronic acid or borate ester (1.5 equiv.), Cu(OTf) 2 (30 mol%), L1 (30 mol%), K 2 CO 3 (4 equiv.), biphenyl (0.02 mmol), DMSO (2 mL), O 2 (3 atm), 140 °C, 6 h. (2) K 2 CO 3 (3 equiv.), MeI (10 equiv.), 25 °C, 12 h. Mechanistic studies were conducted to probe the reaction pathway and based on the product analysis (Figs. 2 d and 3 ), the transformation of lignin to diaryl ether products appears to involve well-ordered C–C and C–O bond cleavage to release reactive phenol intermediates, which subsequently undergo Cham-Lam coupling with the boronic acid. Control experiments employing diol ( c , 1-phenyl-1,2-propandiol), benzyl alcohol ( d ), and acetophenone ( e ) as substrates were conducted to confirm that C α –C β bond cleavage takes place under the reaction conditions (Fig. 5a ). All three different kinds of C α –C β bonds were successfully cleaved affording methyl benzoate in excellent yield (90–98%). These results are in agreement with previous reports concerning Cu catalyzed C–C activation initiated by the oxidation of hydroxy groups (Supplementary Figs. 12 and 13 ) 13 , 15 , 18 , 43 , 44 . In addition, the reaction of protected phenols in which the hydroxy group is modified with glycol ( f ), acetic acid ( g ) and formic acid ( h ) groups with phenyl boronic acid ( 2a ) affords the expected diphenyl ether product in 97–99% yield (Fig. 5b ). Hence, it would appear that under the oxidative conditions the protective groups are unstable 15 , and release reactive phenolic intermediates (Supplementary Fig. 14 ), that react with the boronic acid. Fig. 5 Mechanistic studies. a Control experiments probing C α –C β bond cleavage. Reaction conditions are the same as in Fig. 4 , but without boronic acid. b Control experiments to investigate C(alkyl)−O bond cleavage. Reaction conditions are the same as Fig. 4 . c Exploration of C–C and C–O bond cleavage in β–O–4 lignin model compounds. Reaction conditions were the same as Fig. 4 . d Plausible reaction mechanism. Aromatic dimers with alkyl β–O–4 linkages were employed as lignin model compounds and reacted with 2a under the standard conditions (Fig. 5c ). Dimer i containing a stable methoxy group at the para position provides ester j and diaryl ether 16b in good yield, demonstrating the ability of the catalytic system to degrade the alkyl β–O–4 linkage via C–C and C–O bond cleavage. Alternatively, with a hydroxy group at the para position (dimer k ) diaryl ethers 17b and 16b are obtained, which implies the boronic acid reacts directly with the phenolic hydroxy group. The roles of Cu(OTf) 2 , L1 and K 2 CO 3 were investigated through a series of control experiments (Supplementary Fig. 15 ). The Cu complex and base are indispensable for aerobic −OH group oxidation, C–C bond activation and Cham-Lam coupling. L1 coordinated to the Cu center to promote the Cham-Lam coupling of the boronic acid with the phenol intermediates. The kinetic study demonstrates that C–C bond activation to release reactive phenolic intermediates is slower Cham-Lam coupling between phenol and boronic acid, which ensures rapid capture of phenolic intermediates with boronic acid, preventing side reactions (Supplementary Fig. 16 ). Based on the mechanistic studies a tentative reaction pathway is proposed (Fig. 5d ). Under the basic reaction conditions lignin 1 is activated by the Cu catalyzed oxidation of the hydroxy groups to give intermediate 2 containing a carbonyl group (Supplementary Fig. 12 ) 45 , 46 . Cu catalyzed C α –C β bonds cleavage of 2 results in the depolymerization of lignin to the oxalic acid protected phenol monomer 3 (Supplementary Fig. 13 ) 13 , 18 , 43 , 44 . Thermal decomposition of 3 would release the phenolic hydroxy group to generate reactive intermediate 4 (Supplementary Fig. 14 ) 47 – 49 , which is captured by Cham-Lam coupling with an appropriate boronic acid to afford the carboxylic acid containing product 5 50 , 51 . Methylation of the carboxylic acid in 5 to ester 6 is performed to facilitate analysis. We developed an in -situ method to extract the aromatic rings in lignin by adding aryl boronic acids or borate esters during oxidative decomposition. Diaryl ether products are generated in near-theoretical maximum yield as repolymerization and dearylation side reactions of the reactive phenol intermediates are suppressed. Studies indicate that the reaction is initiated by Cu catalyzed C–C and C–O bond cleavage of the alkyl side chains, degrading the polymeric structure of lignin and releasing reactive phenol intermediates, which are captured by cross-coupling with the aryl boronic acids or borate esters to afford stable diaryl ether products. This study paves the way to alternative approaches to transform lignin via reactive phenol intermediates into high-value specialty chemicals, including pharmaceutical and natural product intermediates and other chemicals."
} | 4,172 |
20161042 | null | s2 | 4,179 | {
"abstract": "We introduce a flow regulating technology that uses trapped air bubbles in a hydrophobic microfluidic channel. We present basic designs for flow regulators and flow valves using trapped air. Experiments have successfully demonstrated the capability of this technique for delivering constant and varying flow rate, and for on-off valving. This approach to valving provides a simple, yet effective way to monolithically integrate flow and valve control on polymer Lab-on-Chip devices."
} | 120 |
29444076 | PMC5812554 | pmc | 4,182 | {
"abstract": "Solitary bees are important but declining wild pollinators. During daily foraging in agricultural landscapes, they encounter a mosaic of patches with nest and foraging habitat and unsuitable matrix. It is insufficiently clear how spatial allocation of nesting and foraging resources and foraging traits of bees affect their daily foraging performance. We investigated potential brood cell construction (as proxy of fitness), number of visited flowers, foraging habitat visitation and foraging distance (pollination proxies) with the model SOLBEE (simulating pollen transport by solitary bees, tested and validated in an earlier study), for landscapes varying in landscape fragmentation and spatial allocation of nesting and foraging resources. Simulated bees varied in body size and nesting preference. We aimed to understand effects of landscape fragmentation and bee traits on bee fitness and the pollination services bees provide, as well as interactions between them, and the general consequences it has to our understanding of the system. This broad scope gives multiple key results. 1) Body size determines fitness more than landscape fragmentation, with large bees building fewer brood cells. High pollen requirements for large bees and the related high time budgets for visiting many flowers may not compensate for faster flight speeds and short handling times on flowers, giving them overall a disadvantage compared to small bees. 2) Nest preference does affect distribution of bees over the landscape, with cavity-nesting bees being restricted to nesting along field edges, which inevitably leads to performance reductions. Fragmentation mitigates this for cavity-nesting bees through increased edge habitat. 3) Landscape fragmentation alone had a relatively small effect on all responses. Instead, the local ratio of nest to foraging habitat affected bee fitness positively through reduced local competition. The spatial coverage of pollination increases steeply in response to this ratio for all bee sizes. The nest to foraging habitat ratio, a strong habitat proxy incorporating fragmentation could be a promising and practical measure for comparing landscape suitability for pollinators. 4) The number of flower visits was hardly affected by resource allocation, but predominantly by bee size. 5) In landscapes with the highest visitation coverage, bees flew least far, suggesting that these pollination proxies are subject to a trade-off between either longer pollen transport distances or a better pollination coverage, linked to how nests are distributed over the landscape rather than being affected by bee size.",
"conclusion": "4.6. Conclusions The model applied in this study is a resource competition model at the time scale of one day, which measures performance parameters at the bee level as proxies for fitness and pollination. Model simulations showed that fragmentation of foraging habitat patches had positive effects on wood-nesting bees, but not on soil-nesting bees. Wood-nesting bees nesting in field edges clump to higher local nest densities and profit from a higher nest to foraging habitat ratio, which increases by fragmentation. This improves fitness and pollination coverage, but decreases pollination distance at the same time. Body size modulated this pattern with smaller bees benefitting more from fragmentation. In terms of traits, large bees have a disadvantage compared to small bees because they have to visit more flower for their pollen requirements (not compensated enough by velocity and short handling time) and wood-nesting bees have a disadvantage because they are limited where they can nest in the landscape and therefore need longer foraging distances. We found that landscape structure clearly affected bees and that improving the ratio of nest to foraging habitat by improving nest opportunities in large fields increases bee fitness and pollination services.",
"introduction": "1. Introduction Wild and solitary bees, important crop pollinators in agriculturally dominated landscapes [ 1 , 2 , 3 ] and essential pollinators of many wild plants [ 4 , 5 ], are clearly declining worldwide [ 6 – 8 ]. Agricultural intensification limits solitary bees to live on resource islands in an unrewarding matrix [ 9 , 10 ], because dominant crops provide hardly any foraging resources (e.g. wheat, maize, rice) and separate nest from foraging habitat [ 11 ], thereby creating a mosaic of fields and natural elements. Such fragmentation at local scales is expected to affect the distribution and pollination potential of solitary bees, which are central-place foragers and therefore prefer foraging resources within several hundred meters from their nest. Natural supply of pollination services by wild pollinators is important for production of many non-dominant crops [ 12 , 13 ] and protecting natural habitats near such crop fields seems to be a key solution to secure it [ 14 , 15 – 17 ]. However, defining how landscape mosaics in agriculturally dominated landscapes can be optimized for wild bees remains a complex subject [ 16 ]. We need to understand how wild bees interact with the landscape to improve landscape configuration and meet all needs of vital wild bee populations. It is clear that wild bee abundance and species diversity at the landscape scale are positively affected by foraging habitat availability [ 18 – 20 ] and nest habitat availability [ 20 – 22 ]. Daily area requirements of solitary bees depend on the distance between nesting and foraging resources [ 11 , 17 ] and hence on landscape fragmentation. Effects of habitat fragmentation, i.e. the process of spatial separation of habitat patches independent of reduced habitat availability [ 23 ], is insufficiently studied for bees and not fully understood. Fragmentation affects bees on at least two different scales. First, fragmentation reduces connectivity of nest sites at larger scales (dozens to hundreds of kilometres) and therefore gene-flow between isolated populations [ 24 ]. At the scale of the agricultural patch mosaic (several hectares to a few kilometers, hereafter termed landscape fragmentation), fragmentation reduces connectivity between nest and foraging sites, affecting daily foraging success. This is of high research interest due to the inevitable consequences for pollination. At the local habitat patch scale (meters), bees may hardly respond to fragmentation [ 25 ]. Habitat fragmentation studies have considered the size of the fragments rather than fragmentation itself [ 26 , 27 , 28 ]. Meadow isolation reduced the number of brood cells of cavity-nesting bees in trap nests [ 29 ]. In spite of a general effect of habitat fragmentation on bees, effects may be species-specific and trait-dependant [ 4 , 9 , 27 ]. The performance of bees in agricultural field mosaics is still difficult to forecast, despite current knowledge on the effect of foraging traits. Two traits are especially relevant for how solitary bee species interact with the landscape and respond to habitat fragmentation: nesting preference and body size [ 30 , 31 ]. Nesting preference determines the home location of their central-place foraging activities, in turn affecting their spatial distribution in the landscape. Body size affects foraging traits such as velocity or capacity for carrying pollen [ 32 ]. The few studies that investigated how wild bee nesting preference and body-size affect responses to fragmentation show contrasting results. Bees nesting above ground are more sensitive to disturbance factors and they are more affected by patch isolation [ 30 ]. At the same time, they can also be more abundant in small patches [ 31 ]. Average body size of wild bee communities was reported to be larger in more isolated [ 33 ] and smaller patches [ 31 ], while also the opposite, relatively more small bees in isolated or small patches has been found [ 28 , 34 ]. Life-history traits, including nesting preference and body size, are often correlated in data sets, obscuring clear effects of single traits [ 30 ]. Our mechanistic understanding of effects of single traits on the way wild bees respond to fragmentation is still incomplete. Small bee species are on the one hand expected to be mostly negatively affected in highly fragmented landscapes, since they may not be able to cross the matrix without foraging resources in agriculturally dominated landscapes [ 21 ]. Bees of intermediate size are expected to be affected when they are mobile enough to leave a large patch, but not mobile enough to reach a distant foraging patch [ 35 ]. On the other hand, all bees may easily survive in a network of patches that are available within their foraging range [ 19 , 36 ], and especially small bees with lower resource requirements may profit. Generally however, bees are good flyers (large more than small) and probably most of them are able to cross an agricultural matrix of hundreds of meters and able to reach distant resource patches. To investigate the performance of different solitary bee types in fragmented landscapes we use the ecologically-detailed model SOLBEE [ 37 ]. SOLBEE is an individual-based, spatially-explicit model to simulate solitary bees foraging for pollen in the landscape. Individual-based or agent-based models (IBM/ABM) are a well-established approach to investigate competition and resource depletion in space and time, such as solitary bees that adapt their movement to local changes made by other solitary bees. SOLBEE incorporates behaviour and decision-making known for solitary bees and allows investigation of species traits (e.g. body size and nesting preference). The model is a resource competition model during one day in a 100 ha landscape, which measures performance parameters at the bee level as proxies for fitness and pollination. In this study we investigate small-scale fragmentation in landscape mosaics (scale of one kilometre), where foraging resources and fragmented edge habitat affect daily foraging performance and pollination services. Bees differing in body size and nesting preference are expected to differ in their performance. Our goal is to gain a better understanding of how different bees cope with local spatial resource distribution and how spatial pollination is affected. Specifically we ask: What is the effect of landscape fragmentation on daily performance, and can we observe both the negative and positive effects as shown by field studies? How does local landscape fragmentation affect pollination services? Do different bee types (differing in body size and nesting preference) respond differently to landscape fragmentation? Do different bee types provide pollination services differently? Do pollination proxies provide a consistent pattern or are there trade-offs? Can we define optimal conditions in fragmented landscapes for 1) bee fitness (daily performance of solitary bees) and 2) pollination, which 3) can be generalized for different bee types?",
"discussion": "4. Discussion In all simulations both large and small bees could, based on resource availability at the landscape scale, build the same number of brood cells and cover the complete foraging habitat. Thus, all observed differences in brood cell numbers (and pollination proxies) between soil- and wood-nesting bees and bees of different size is due to the different allocation of foraging and nesting resources, especially the latter being affected by fragmentation. The limited amount of time (a day) and the time budgets related to body size have resulted in differences. 4.1. Effects of spatial resource distribution 4.1.1. Fragmentation effects on fitness By design, all bees had the opportunity to build the same number of brood cells within a day assuming spatial allocation of nests and pollen producing flowers would not play a major role. This was indeed the case for soil-nesting bees of the same size, which did not respond to landscape fragmentation ( Fig 2a ) or to the ratio of nest to foraging habitat ( Fig 3c ). They had optimal access to foraging resources from their nest, because nests were distributed randomly over the foraging habitat ( Fig 1c ). Wood-nesting bees, in contrast, responded positively to landscape fragmentation . This supports the hypothesis that fragmented landscapes increase bees diversity by providing increased nest-site availability [ 26 , 50 ], which applies to wood-nesting bees restricted to edge habitat. In our model, bees were indeed positively affected by nest-habitat availability ( Fig 3a ). Although the interaction effect between foraging habitat availability and landscape fragmentation was low, wood-nesting bees seem to respond stronger in landscapes with high foraging habitat availability ( Fig 2a ). The apparent decrease in brood cell number with increasing foraging habitat availability ( Fig 2a ) is a side-effect of the model setup, highlighting the importance of time and time-budgets. The number of individuals per landscape was by definition linked to foraging habitat availability . The resulting local bee density (and hence competition around nest sites for resources) had a strong negative effect on brood cell number ( Fig 3b ). This supports the idea that time can be more limiting for wild bees than foraging resources [ 47 ]. Spatially induced time constraints deserve more attention in pollinator research. It can for example be argued that oligolectic and monolectic bees (highly specialized on certain plant species, e.g. Andrena hattorfiana [ 51 ]) face very low plant densities at the landscape scale and have to deal with strong time constraints, an additional competitive disadvantage compared to polylectic bees. We showed that traits ( nesting preference and body length ) affect the response to fragmentation, explaining why some studies find no effects of fragmentation on bee performance [ 20 , 26 ], while others did [ 19 , 27 ]. Lack of response to fragmentation in field studies could relate to dominance of certain traits in the community (e.g. a large proportion of bumblebees). Nevertheless, we also recognize that the effect of landscape fragmentation can be low compared to other landscape-level parameters (compare Tables 3 and 4 ) and may remain undetectable in many field studies, suggesting a need for alternative measures. Finally, also an increase in foraging habitat availability above an intermediate amount reduced the amount of edge structures (merging of patches), which is independent of landscape fragmentation (e.g. nest habitat availability is frequently low beyond low landscape fragmentation , Fig 3a ), again demonstrating that landscape fragmentation on its own is a poor measure. 4.1.2. Fragmentation effects on pollination The pollination measures (number of flower visits, percentage visited foraging habitat and mean foraging distance) largely followed the patterns for the number of brood cells. Pollination by soil-nesting bees remained largely unaffected by fragmentation. However, the pollination performance of wood-nesting bees was affected by fragmentation. Wood-nesting bees visited fewer flowers ( Fig 2b , small and intermediate sized wood-nesting bees) and flew shorter distances ( Fig 2e ) with increasing landscape fragmentation . Under high local bee density (low landscape fragmentation ), bees inevitably encountered empty flowers more frequently and were forced to visit and probe more flowers ( Fig 3e ) and fly longer distances ( Fig 3k ). This means that fragmentation can both reduce the frequency of pollen transfer between flowers and reduce the mean distance over which the pollen is transferred. Landscape fragmentation increased the landscape-level coverage with pollinators (percentage visited foraging habitat, Fig 2c ) by a better distribution of nest habitat over the landscape. 4.1.3. Linking fragmentation, nest habitat, bee density and the ratio of nest to foraging habitat Increased landscape fragmentation resulted in higher nest availability, a lower local bee density and a higher ratio of nest to foraging habitat. Local bee density and the ratio of nest to foraging habitat were both better predictors for brood cells, visited foraging habitat and foraging distance than landscape fragmentation itself. The nest to foraging habitat seems a suitable landscape-level parameter to replace landscape fragmentation , foraging habitat availability and nesting preference by a single measure, giving an informative gradient of habitat complementarity for wild bees [ 11 ], capturing local landscape quality well ( Fig 3c, 3f, 3i and 3l ). Although local bee density was an equally good predictor—and can in principle be measured in the field—it is less practicable because it requires high sampling effort. The ratio is probably easier to approximate with local information about potential bee habitats. The result that bees need an appropriate ratio of nest habitat relative to foraging habitat can be considered a novel, but also logical, insight gained by modelling practice. We believe that the general observation that wild bee communities are mainly affected by foraging habitat availability [ 18 – 20 ] is therefore incomplete. The hypothesis that solitary bees are most limited by nest sites [ 20 – 22 ] applies therefore, according to our results, only when foraging resources are constant. The here proposed ratio of nest to foraging habitat availability could satisfy the need for a landscape measure that is more suitable in describing the resource needs of solitary bees [ 36 ]. 4.2. Effect of traits on spatial foraging 4.2.1. Body size One would expect a large bee to visit more flowers (required for a full load) and fly longer distances than a small bee. This was in general true ( Fig 2b and 2d ). As a result, large bees built fewer brood cells than small bees, which is also supported by field observations (reviewed in [ 37 ]). The expectation that large bees perform better than small bees in fragmented landscapes because of their faster movement [ 50 ] and shorter flower handling times, turns out to be wrong, at least under the assumptions made. The number of visited flowers was the response most affected by body length . Obviously, large bees need to visit more flowers for their pollen requirements than small bees ( Fig 2b ), in accordance with field studies [ 52 , 53 ]. The difference in flower requirements per brood cell between large and small bees is even higher than the difference in daily requirements, given that large bees also build fewer brood cells. Time allocation diagrams for the different bees ( S2 Appendix Fig B) make clear that model bees spent far more time visiting flowers than for flying between them or back to the nest. Therefore, flower-handling time is the most important trait for compensating high pollen requirements. This trait was in the sensitivity analysis indeed the most influential one among other size-related traits [ 37 ]. When flowers are larger, the explained variance by body length for brood cells drops more than half, the most prominent difference for altered vegetation parameters ( S3 Appendix Table A). Larger flowers bring an advantage for large bees when they do not have to visit so much more flowers than smaller bees (indeed reduced explained variance by body length for flower visits, S3 Appendix Table A). Small bees have an advantage compared to large bees, but they are also more sensitive to changes in the landscape. Small and intermediate-sized bees (wood nesting) visited fewer flowers with increasing landscape fragmentation , while large bees (wood nesting) visited more flowers ( Fig 2b ). In addition, the crossing lines in Fig 3c show that there are also landscapes in which small bees perform worse than larger bees. In landscapes with much foraging habitat and little nest habitat (low nest to foraging habitat ratio, Fig 3c ) local nest density of bees is very high with small bees having the highest nest densities ( Fig 3b ). This nest density problem is most problematic for small bees due to the mass proportional relation between body size and bee numbers in the landscape ( S1 Appendix Table D). At some point the density of small bees becomes so large that flying farther from the nest for foraging resources ( Fig 3k ) cannot compensate this, leading to a very steep decrease of brood cell number which is absent for large bees ( Fig 3b ). Also does an increase in local bee density imply more visits of empty flowers, causing an increase of total flower visits for small bees, while large bees show a slight decrease ( Fig 2e ), explaining the different responses to landscape fragmentation (which reduces local bee density). Large bees seem to escape local overpopulation more easily (steeper increase in mean foraging distance with local bee density, Fig 3k ). Consequently, especially small bees could be driven to evolve mechanisms for optimizing their foraging behaviour and to quickly sense high local bee densities and empty flowers (e.g. by smart flower probing rules), to justify the investment in farther flights. Larger bees seem in general to be better pollinators: they visit more flowers, cover more foraging habitat and fly longer distances. For visited foraging habitat is the effect of body size lowest, and small bees cover the foraging habitat almost as much as large bees ( Fig 3g–3i ) although they fly on average less far from the nest ( Fig 3j–3l ). The pollination potential of small insects is often underestimated and they transfer enough pollen for sufficient seed set [ 54 ]. 4.2.2. Nesting preference Nesting preference was the most important predictor for foraging distance and for visited foraging habitat and second important for the number of brood cells. Soil-nesting bees were evenly distributed over the foraging habitat and found foraging resources near the nest, leading to short foraging distances ( Fig 2d ) and optimal coverage of the foraging habitat ( Fig 2c ). The maximum number of brood cells within a day ( Fig 2a ) was hardly affected by the gradient of landscape fragmentation . Wood-nesting bees, in contrast, responded strongly to different landscapes. They had a lower brood cell number in landscapes with a low degree of landscape fragmentation ( Fig 2a ) where they had longer foraging distances ( Fig 2d ). This makes sense in relation to lower nest habitat availability ( Fig 3a ), higher local bee density ( Fig 3b ) and low ratio of nest to foraging habitat availability ( Fig 3c ). Wood-nesting bees also covered less foraging habitat when there was more foraging habitat to cover in the landscape (down as low as 25% for high foraging habitat availability , Fig 2c ). Bees nesting at field edges did not reach the interior of the fields in those cases. Nesting preference hardly affected the number of flower visits ( Fig 2b ), which seems rather being affected by body-size related traits alone. Soil and wood nesting is often considered as a factorial contrast between bees, but the model shows otherwise. There seems to be a gradient of habitat use, where both nesting preferences are part of the same relationship for a bee of a certain size ( Fig 3 , all panels). Most models explained the variance sufficiently without nesting preference ( Table 4 ). Soil-nesting bees in natural bee communities often nest in very high densities [ 55 ] and occur in much higher numbers on fallow land than wood-nesting bees [ 19 ], suggesting that soil-nesting should be modelled more restricted in nest habitat availability. This would likely complete the visible gap (especially in the ratio of nest to foraging habitat, Fig 3c, 3f, 3i and 3l ). 4.3. Pollination considerations 4.3.1. Trade-offs between pollination measures We found a trade-off between mean foraging distance and coverage of the habitat with pollinators at the landscape level (negative relationship, Fig 4c and 4d ). This means that either pollen is transported over larger distance or that pollen is transported at all places in the foraging habitat, not both. In the best landscapes for bees (e.g. high ratio of nest to foraging habitat), bees were spatially optimally distributed leading to a high percentage of visited foraging habitat ( Fig 3i ), also leading low mean foraging distances ( Fig 3l ). Oppositely in bad landscapes, bees are forced to fly farther distances, but do not cover all foraging habitat remaining certain areas unpollinated. In agricultural landscapes it is common that bees are most abundant in field edges [ 56 , 57 ]. Field interiors often show low abundance of solitary bees, and bees foraging there may be soil-nesting bees provided locally with nests [ 58 ]. 4.3.2. Up to 20% suitable as nesting habitat The simulation results suggest that visited foraging habitat (coverage with bees) increases steeply up to a nest to foraging habitat ratio of 0.2 (i.e. 20% nest habitat compared to foraging habitat) and beyond this levels off to optimal coverage (close to 100%, Fig 4a ). The value seems robust for vegetation type ( Fig 4a and 4b ) and bee type. It may suggest a general pattern applying to bees as pollinators in general, but needs deeper investigation and field validation for broader application. This value reminds of the recommendation that about 25% of the landscape should remain refugee area for wild bees to maintain sustainability [ 59 ]. A recent review [ 60 ] estimates that 2 to 44% of the landscape with high quality flower strips is required for the sustainability of pollinator communities. However, those numbers focus on a different aspect of the bees' ecology (sustainable populations) and do not consider the required amount of nest habitat (and ratio of bee habitats) to pollinate a certain area. Hence, data-based estimations of the optimal ratio remain a future concern. A simple calculation illustrates that most agriculturally dominated landscapes are really poor and a hostile environment for solitary bees, despite mass flowering crops. For example, providing a one ha crop field with a five-meter wide natural strip yields a desired ratio of 0.19. The same five-meter strip at a 49 ha field yields a ratio of 0.03. Practically considered, to improve the local ratio, the nest habitat for bees should be sufficient (increase of unmanaged field strips) of good quality (reduced pesticide application) and spatially well distributed within an area (fragmented spatial distribution). We recognize that 20% field strips is not economically feasible in modern agriculture, but we agree with others that management encouraging a mosaic of smaller fields and increase of the edge:area ratio would benefit pollinators [ 61 ]. For example, up to 8% of the field edge can be converted to bee-friendly habitat without losing yields [ 62 ], because the positive effect on pollinators (increased pollination) compensates for the smaller crop area. In situations where the ratio remains far below 0.2 (e.g. large crop fields), a solution may be the provision of artificial nest sites. There are many examples where solitary cavity-nesting bees are employed as crop pollinators, by offering artificial nests [ 63 , 64 ]. Also, at least one soil-nesting species ( Nomia melanderi ) is managed as crop pollinator by offering nesting beds in the soil [ 65 , 66 ]. 4.4. General considerations 4.4.1. Foraging distances The mean foraging distance from the nest is a measure for how far pollen is transported and it is focused on the motivation of the bee. Assuming that bees do not fly farther than necessary can result in very short foraging distances for soil-nesting bees (< 50 m, Fig 2d ) in accordance with natural ranges from field studies [ 37 ]. Their nests were evenly distributed over the foraging habitat and they found enough pollen within a short range from the nest in all landscapes. Mean foraging distances are higher when conditions are unfavourable, e.g. when bees face a high local nest and bee density in field edges (wood-nesting bees) and untouched foraging resources only farther away. Large bees flew farther (50–200 m) than small bees (30–100 m). These still relatively low distances are in agreement with the finding that that both large and small bees often forage below 200 m [ 67 ]. Some studies may overestimate foraging distances, especially when solitary and eusocial bees are not separated or landscape types are not considered [ 16 ]. Honeybees and bumblebees fly farther, as well as solitary bees without any foraging recourse near the nest. The mean foraging distance as measure for pollination does not provide information on extreme events (beyond the mean) that may lead to pollination as well. Nevertheless, it is clear that bees mainly exchange pollen between plants over short distances and that most activity is near the nest. 4.4.2. Matrix crossing Short foraging distances imply that bees did not often fly to more distance patches and did not often cross the matrix without foraging resources, despite a parameter that induces such behaviour. Matrix crossing behaviour was modulated by the model parameter ignorance ( Table 1 ), which was earlier shown not to affect the model simulations much [ 37 ]. Low matrix crossing behaviour is in accordance with the idea that wild bees live on islands of foraging habitat [ 9 ] and is reported for various pollinators [ 68 , 69 ]. Solitary bees have a high site fidelity with very conservative movement patterns [ 70 ], which does not seem to be improved by grassy field strip corridors [ 57 ]. The idea that solitary bees, as flying insects, often display matrix crossing behaviour and fly long distances needs to be further refined by future field and modelling studies. In ecosystem-service research, such concepts are trivial for estimating the spatial availability of pollination services. 4.4.3. Size and evolution The result that large solitary bees are on average worse performers than small ones may imply evolutionary consequences. The lower efficiency of large bees may be a driver to develop a social structure with higher efficiency and explain why large bees are more often social than small bees. In central Europe there are e.g. more species of eusocial bumblebees than large bees from the genus Xylocopa or Anthophora , in contrast to very small bees which are often solitary (e.g. Andrena ) or only primitively eusocial (e.g. Lasioglossum ). The performance constraint may thus be an additional driver for sociality in combination with other evolutionary drivers such as climate change [ 71 ] and time (lineage age [ 72 ]). At the same time it may explain why in regions where bees have been under pressure for decades, body size decreased over time at the species level [ 73 ]. 4.4.4. Ratio of habitats as simplified measure Foraging habitat visitation increased with the ratio of nest to foraging habitat with a remarkably low effect of body length . This ratio seems a robust proxy descriptor for landscape structure, adapted to the perspective of bees and suitable for estimating pollination services. We expect that the ratio of nest to foraging habitat, after further study, can become an easy to calculate landscape simplification in field studies and a valuable addition to other indexes such as the LLI [ 74 , 75 ]. When more field surveys include the identification of nest habitat in addition to foraging habitat, we expect that the positive effects of the ratio can be soon be confirmed. 4.5. Future considerations 4.5.1. Notes on biological assumptions in the model The combined fact that we tested all model parameters [ 37 ], that the model's output values come close to values from real systems ( Fig 3 ), and that we ensured that our results hold for altered vegetation parameters, confirms that we have successfully covered a part of a complex multifactorial system and understand this part a little better. As with most models, some biological assumptions lie in the intrinsic structure of the model itself and are not critically tested, while at the same time simplifications are inevitable to speed up calculation. Some of our decisions and considerations should be mentioned for future model implementations. One example is the unrealistically high number of brood cells, which can easily be justified but also criticized. We assumed a short time at the nest after each trip and we neglected time needed for egg laying, cell closure and other activities (due to data deficiency and unnecessary complexity, see also [ 37 ] for a discussion). We further assumed that all pollen collected at the flowers reach the nest, while in reality pollen gets lost on the way [ 76 ] and solitary bees of the same species return to the nest with a high variability of pollen loads [ 77 ]. Also, in favourable landscapes bees may be egg-limited rather than pollen limited [ 38 ]. Some assumptions may have excluded important mechanisms from the model, such as the assumption that bees also fuel themselves with nectar on the same flowers when they forage for pollen. The time budget for nectar collection is relatively small compared to that for pollen collection (see [ 37 ]) and may be negligible, but we do not know the effect of reducing a decision sequence depending on two resource levels to a single one. Finally, considering the many different parameters that were parameterized with heterogeneous sources, the model may benefit systematic studies for each parameter to improve accuracy. To identify priorities, a future study could address how changes in the allometric rules affect time budgets ( Table 2 ) and identify the rule requiring the highest quality data. Also the effect of potential correlations, such as a positive relation between body size and flower size preference (which probably would minimize the disadvantage for large bees that we found) or a positive relation between body size and time at the nest , could be of future interest, even when studies failed to prove a systematic difference in flower preference [ 78 , 79 ] or time at the nest (reviewed in [ 37 ]) so far. In the end, the priory of considering such issues depends on the questions for which the model is used. 4.5.2. Towards simulation of real communities We chose to focus on fragmentation, kept other parameters constant and compared six bee types in a scenario-like way to reduce complexity and understand model processes. As may be desired by field ecologist working with pollinators, simulation of realistic plant and bee communities requires a data-driven approach, in which values are parameterized and combined in sets. For a simulation with multiple bee species one needs community data on species composition, local bee densities and nesting locations. It is already challenging to get such sufficiently detailed data in the field, but the model also requires definition of flower traits making up the vegetation, including pollen provision and flower density. As a minimum, the distribution of these traits in a community is needed to simulate realistic vegetation patches. At the same time, additional model rules may be required when bee species face different types of flowers or when the presence of other species affects foraging behaviour [ 80 ] or brood cell number [ 81 ]. A further advantage of such an attempt with real bee densities for each species separately is that it overrules the assumption that pollinator density scales negatively with body size and positively with foraging resources, which had a prominent effect on our results. 4.5.3. Applications with crop fields and semi-natural edge habitats The model system may roughly represent crop systems: fields with foraging resources and edge habitat with nesting resources for wood-nesting bees (and in some cases suitable soil within the crop field). However each crop has different properties that are not necessarily covered by our current simulations. Flower size (pollen production per flower) and flower density can be much more extreme, such as a clover field with very small flowers in high densities or a sunflower field with very large flowers in low densities. We did not model this explicitly for several reasons. We focussed on a more general system to understand wild bees as pollinators and general patterns, but specific crops systems could be focus of future studies. The current results help to think in terms of flower sizes and densities in real systems and to interpret other studies in this light. For example, in a recent study wild bees were hardly found in sunflower field interiors [ 82 ], which may be a result of enough pollen being offered near the nest, explaining that the quality of the edge habitat did not have an effect on the pollinator community in the interior of the field [ 82 ]. Our model suggests that an increase in fragmentation and the ratio of nest to foraging habitat at a very local scale should increase pollinator coverage largely independent of flower size. A different challenge for the future is to see and treat an agricultural field as potential nest habitat for wild bees. When soil-nesting bees are limited to field edges they must fly farther distances from the nest and face more local competition with bees around the nest. In general, soil disturbance in crop fields is assumed to be too high for soil-nesting bees to survive [ 83 – 85 ], but recent studies suggest that it is possible [ 58 , 86 ] for species that nest a meter below the surface. Agricultural practices that reduce the depth of mechanical 'action' and pesticide application will benefit the survival of soil-nesting bees within fields. This would give an optimal nest to foraging habitat ratio and lead to better pollination. 4.5.4. Combining strengths of pollinator models Several models with foraging bees in response to vegetation seem to have being developed in parallel [ 37 , 87 – 90 ]. Pollination ecology and agricultural ecology could benefit from a kind of \"master-model\" in which strengths are combined. This requires important decisions on how to deal with different scales (requiring different model elements) and reduce complexity in favour of simulation time. We think that some elements in our model that are unpractical in application can be simplified. For example, our decision to use mass-scaled resource availability for each bee (resulting in many small bees being compared to a few large bees), is not very practical to combine with an IBM approach, since a high variance in individual numbers also results in a high variation in calculation times. Also, some of our parameters are elaborate to measure and may benefit clever proxies. Presently, each model has its own advantages and level of detail, applicable to selected research questions. 4.6. Conclusions The model applied in this study is a resource competition model at the time scale of one day, which measures performance parameters at the bee level as proxies for fitness and pollination. Model simulations showed that fragmentation of foraging habitat patches had positive effects on wood-nesting bees, but not on soil-nesting bees. Wood-nesting bees nesting in field edges clump to higher local nest densities and profit from a higher nest to foraging habitat ratio, which increases by fragmentation. This improves fitness and pollination coverage, but decreases pollination distance at the same time. Body size modulated this pattern with smaller bees benefitting more from fragmentation. In terms of traits, large bees have a disadvantage compared to small bees because they have to visit more flower for their pollen requirements (not compensated enough by velocity and short handling time) and wood-nesting bees have a disadvantage because they are limited where they can nest in the landscape and therefore need longer foraging distances. We found that landscape structure clearly affected bees and that improving the ratio of nest to foraging habitat by improving nest opportunities in large fields increases bee fitness and pollination services."
} | 10,006 |
36668687 | PMC10106850 | pmc | 4,183 | {
"abstract": "Summary Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large‐scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five‐year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2‐oxoglutarate‐dependent dioxygenase ( 2OGD ). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE , ( SuSy ), CINNAMATE 4‐HYDROXYLASE ( C4H ) and increased levels of GTPase activating protein for ADP‐ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild‐type. The pilot‐scale industrial‐like pretreatment of best‐performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.",
"conclusion": "Conclusions This study evaluated seven constructs represented by 32 transgenic lines pre‐selected in large‐scale greenhouse screening by determining their growth and wood properties relevant for saccharification after five‐year cultivation in the field. The results (i) provided relevant information on field testing strategies, (ii) revealed physiological functions of tested genes in natural environment, (iii) identified types of transgenic manipulations suitable for saccharification improvement and (iv) revealed relationships among different traits and saccharification, identifying key parameters governing better saccharification yields. Moreover, the best transgenic lines were processed in a pilot‐scale reactor under industrial‐like conditions, providing proof‐of‐concept results supporting our strategy of developing biorefinery‐improved feedstocks.",
"introduction": "Introduction Wood is the most abundant, naturally degradable and renewable carbon source on Earth (Bar‐On et al ., 2018 ), and technologies are currently being developed allowing its complete utilization. In biochemical conversion, pretreatment is used as an initial fractionation facilitating enzymatic saccharification of cellulose prior to microbial fermentation of sugars and recovery of hydrolysis lignin (Martín et al ., 2022 ). However, the recalcitrance of wood hampers its biochemical conversion, which is problematic considering that mild reaction conditions with low input of chemicals and energy, and high product yields are important for sustainable and competitive processes (Li et al ., 2014 ; Martín et al ., 2022 ). Thus, overcoming recalcitrance and achieving high sugar yields are main priorities in developing new generation biorefinery feedstocks. Hardwood species including fast‐growing poplars or aspens are well suited for biochemical conversion provided that their recalcitrance is reduced (Hinchee et al ., 2009 ; Ko et al ., 2020 ; Kumar and Verma, 2021 ; Sannigrahi et al ., 2010 ). Recalcitrance is a multiscale phenomenon, related not only to the composition and molecular structure of the different lignocellulosic components but also to their supramolecular interactions and hierarchical organization (Himmel et al ., 2007 ; McCann and Carpita, 2015 ; Silveira et al ., 2013 ). It could be reduced by breeding, which is a slow process in trees, or by more direct genetic engineering of wood (Chanoca et al ., 2019 ; Donev et al ., 2018 ). There are many examples of recalcitrance reduction by genetic engineering (Biswal et al ., 2014 , 2015 ; Eudes et al ., 2012 ; Gandla et al ., 2015 ; Hao et al ., 2021 ; Macaya‐Sanz et al ., 2017 ; Park et al ., 2004 ; Pawar et al ., 2017 ; Wilkerson et al ., 2014 ). In some cases, the beneficial impact on enzymatic saccharification comes at the expense of growth and survival (Gandla et al ., 2015 ; Van Acker et al ., 2014 ), which calls for better understanding of plant traits related to saccharification. Moreover, in most cases, transgenic plants are tested in the greenhouse experiments. It is however recognized that novel off‐target effects can be observed in genetically engineered trees in the field (Derba‐Maceluch et al ., 2020 ; Funahashi et al ., 2014 ; Pramod et al ., 2021 ; Strauss et al ., 2016 ; Taniguchi et al ., 2012 ). Therefore, there is a need to generate more field experimental data to identify promising genetic engineering strategies that improve tree productivity and wood properties in woody feedstocks designated for biochemical conversion. The aim of this study was to evaluate the productivity and wood properties related to biochemical conversion of field‐grown transgenic hybrid aspen ( Populus tremula L. × tremuloides Michx.) lines that were selected by extensive greenhouse screening and are part of a large collections of transgenic lines generated to understand and characterize the functions of genes highly expressed in wood‐forming tissues (Bjurhager et al ., 2010 ; Escamez et al ., 2017 ; Gerber et al ., 2014 ). Selected constructs targeted genes encoding enzymes with known or predicted functions as well genes which functions have not yet been characterized (Table 1 ). The recalcitrance of analysed genotypes was assessed by analytical saccharification with and without acid pretreatment (Gandla et al ., 2015 ), nanoporosity (Wang et al ., 2019) and subcritical water extractability (Martínez‐Abad et al ., 2018 ). Further, we have explored relationships between 49 different traits reflecting productivity, wood quality and saccharification efficiency and built predictive models for saccharification yields. The best construct was tested in a pilot‐scale pretreatment followed by analytical saccharification, to confirm its superiority in industrially relevant conditions. The study evaluates the benefits of transgenic manipulation of known and unknown genes and gives invaluable information on the importance of different traits in natural field conditions for saccharification outputs. This information can be used in tree genetic engineering as well as in tree breeding aiming at reducing recalcitrance of wood. Table 1 List of constructs used in this study Cons‐truct Type \n P . trichocarpa gene ID ( v3 . 1 ) \n P . tremula gene ID ( v2 . 2 ) \n A . thaliana ortholog Activity Function References UAP RNAi Potri.003G074700 Potra2n3c7862 \n AT2G35020 \n GLCNA.UT2 \n UDP‐N‐acetylglucosamine pyrophosphorylase Synthesis of glycoproteins and glycolipids Yang et al ., ( 2010 ), Chen et al . ( 2014 ) SuSy RNAi Potri.006G136700, SUS1 Potra2n6c14105 \n AT3G43190 \n SUS4 \n Sucrose synthase Cell wall polymer biosynthesis Gerber et al . ( 2014 ), Dominguez et al . ( 2021 ) C4H RNAi Potri.013G157900 Potra2n13c24959 \n AT2G30490 \n C4H \n Cinnamate 4‐hydroxylase Lignin biosynthesis Bjurhager et al . ( 2010 ), Sewalt et al . ( 1997 ) SAM RNAi Potri.014G114700 Potra2n14c27264 \n AT4G01850 \n SAM2 \n S‐adenosylmethionine synthetase General methylation reactions Jin et al . ( 2017 ) 2OGD RNAi Potri.009G107600 Potra2n9c19173 AT3G19000 2‐Oxoglutarate‐dependent dioxygenase family Unknown MYBL OE LMP1 ‐promoter Potri.006G000800 Potra2n6c15386 AT2G01060 MYB‐like HTH transcriptional regulator Unknown ZAC OE 35 S ‐promoter Potri.001G372000 Potra2n1c3260 \n AT4G21160 \n ZAC/AGD‐12 \n ARF‐GTPase‐activating protein Vesicle trafficking Jensen et al . ( 2000 )",
"discussion": "Discussion Line performance changes after the first year in the field In this study, 32 transgenic lines belonging to seven constructs were tested over five years in the field trial. The targeted genes represented variety of functions and were upregulated during secondary wall formation in developing xylem (Figure 1 ). All tested lines showed changes in target gene expression in the developing wood of field‐grown trees (Figure 1 ) consistent with previous greenhouse analyses (Bjurhager et al ., 2010 ; Gerber et al ., 2014 ), supporting the long‐term stability of transgene expression in the field reported previously (Hawkins et al ., 2003 ; Pramod et al ., 2021 ; Strauss et al ., 2016 ). However, line growth performance in the field was in some cases different than in the greenhouse (Figure 2 ; Figure S1 ; Bjurhager et al ., 2010 ; Gerber et al ., 2014 ). A major re‐ordering of transgenic lines with regard to several parameters occurred after the first dormant period (Figure 3b ) indicating that the overwintering is a critical test of transgenic trees (Strauss et al ., 2016 ). This could be related to several reasons: (i) transgene expression could interfere with physiological processes during overwintering; (ii) transgene insertion could disrupt genes having a critical role for overwintering and (iii) transgene insertion could interfere with genomic rearrangements that occurs in meristems of overwintering trees (Lloyd et al ., 1996 ; Mellerowicz et al ., 1989 , 1992 ). Thus in boreal climate, at least a two‐year testing period is needed to evaluate growth of transgenic perennial crops and results from shorter duration field studies (Kim et al ., 2018 ; Macaya‐Sanz et al ., 2017 ) should be treated with caution. Function of targeted genes revealed by field testing \n SuSy encoded sucrose synthase provides UDP‐Glc from sucrose for cellulose and other cell wall polymers (Stein and Granot, 2019 ), and it is proposed as a key enzyme regulating wood cellulose biosynthesis (Coleman et al ., 2009 ). In contrast to mild effects of SuSy downregulation in the greenhouse (Gerber et al ., 2014 ), which were explained by an alternative route of UDP‐Glc biosynthesis mediated by invertase (Dominguez et al ., 2021 ), the growth of SuSy ‐suppressed lines was greatly reduced in the field (Figure 2 ; Dominguez et al ., 2021 ). Moreover, their wood displayed anatomical abnormalities, reduced cell wall thickness and density, reduced carbohydrate and increased lignin contents and its sugar composition indicated a reduction in cellulose compensated by higher hemicellulose and pectin contents (Figures 3 and 4 ; Figures [Link] , [Link] ). These observations indicate that SuSy ‐encoded activity mediates C‐flux not uniformly to all wall components but primarily to cellulose, which is needed for cambial activity and secondary wall biosynthesis in developing xylem, and which cannot be efficiently substituted by any alternative pathway in the field conditions. \n C4H encodes a cinnamate 4‐hydroxylase involved in the conversion of t ‐cinnamic acid to p ‐coumaric acid (Boerjan et al ., 2003 ; El Houari et al ., 2021a ). Mutation in C4H in Arabidopsis thaliana reduces lignin content and induces growth abnormalities (Schilmiller et al ., 2009 ) that were recently shown to be caused by accumulation of c ‐cinnamic acid interfering with polar auxin transport (El Houari et al ., 2021b ). C4H downregulation in aspen also induced dwarfism and anatomical wood abnormalities, reduced lignin and increased phenolics content (Figures 2 , 3 , 4 ; Figures S2 and S3 ; Bjurhager et al ., 2010 ), consistent with reports in A . thaliana (Boerjan et al ., 2003 ; El Houari et al ., 2021a ). \n Arabidopsis thaliana ZAC encodes a protein activating GTPase activity of arabinosylation factors (ARFs) involved in endosomal vesicle trafficking (Jensen et al ., 2000 ) and mediating gravitropic bending in response to Ca +2 fluxes (Dümmer et al ., 2016 ). In aspen, ZAC is broadly expressed in wood‐forming tissues (Figure 1 ), and its upregulation strongly inhibits height and diameter growth (Figure 2 ) and affects wood cell structure (Figures S2 and S3 ). These results indicate that ZAC has an essential cellular function for cell division, radial cell expansion and secondary wall thickening. \n SAM is predicted to encode a methionine adenosyltransferase – an essential enzyme synthesizing S‐adenosylmethionine that serves as general methyl donor in a variety of reactions including lignin and ethylene biosynthesis (Jin et al ., 2017 ). SAM downregulation in aspen affected tree growth with different outcomes; a strong downregulation inhibited height and diameter growth, but a mild downregulation stimulated it (Figure 2 ), but there were no changes in lignin content even in the most suppressed line (Figure 4 ). As the suppression of two SAM homologues in switchgrass resulted in growth inhibition and strong lignin reduction (Li et al ., 2022 ), we hypothesize that the lack of similar effect on lignin in aspen is due to the redundancy in the gene family. It would be also interesting to investigate if the nonlinear growth response of transgenic lines to SAM transcript dosage could be related to an epigenetic effect via DNA and histone methylation known to be regulated by the methionine level (Yan et al ., 2019 ). Aspen 2OGD is a member of a large family of 2‐oxoglutarate‐dependent dioxygenases, which couple two‐electron oxidation of diverse substrates with decarboxylation of 2‐oxoglutarate to succinate. The family has three subclades (Kawai et al ., 2014 ). 2OGD belongs to the subclade DOXC that includes oxygenases involved in biosynthesis of gibberellins, flavonoids and ethylene, but the branch 21 of this subclade where 2OGD belongs has not yet been functionally characterized. In wood‐forming tissues of aspen, 2OGD is expressed in cells depositing secondary walls (Figure 1 ), and its suppression tended to stimulate stem height growth and significantly increased stem diameter and biomass in the most suppressed line (Figure 2 ), suggesting that activity of 2OGD inhibits stem primary and secondary growth. Constructs with less recalcitrant biomass The C4H construct had greatly improved Glc yield and conversion in NP (Figure 5 ; Figure S6 ), but these positive changes were offset by growth penalty and abnormalities in wood development. The results are similar to those obtained for C4H ‐downregulated hybrid eucalyptus grown in the field for 2 years (Sykes et al ., 2015 ) and field‐grown transgenic Populus with downregulated different monolignol biosynthetic pathway genes (Pilate et al ., 2002 ; Voelker et al ., 2011 ; Van Acker et al ., 2014 ). These results show that growth inhibition and developmental abnormalities are observed when the lignin content is significantly reduced, which limits the use of lignin‐reduced transgenic trees. However, recent discoveries in lignin‐downregulated Arabidopsis and Medicago showing that developmental abnormalities had indirectly been induced by signalling involving polygalacturonase or bioactive phenolic compounds (El Houari et al ., 2021b ; Gallego‐Giraldo et al ., 2011 , 2020 ) open possibilities to develop strategies that control the signalling of lignin deficiency. C4H plants had several interesting characteristics from the biorefinery point of view, like a lower S/G ratio, a higher carbohydrate content and nanoporosity (Figures 4 and 7 ; Figures S5 and S7 ), and their extracts were enriched in high molecular weight compounds (Figure 8 ), providing incentive to develop a remedy for the growth defect. \n SuSy ‐downregulated lines had highly improved Glc yields and conversion in PT (Figure 5 ; Figure S6 ). This interestingly occurred despite their increased S‐lignin contents (Figure 4 ), supporting the efficiency of acid pretreatment in S‐lignin modification such, that its presence does not limit cellulose enzymatic conversion anymore (Li et al ., 2016 ). Increased saccharification can be explained by increased nano‐ and microporosity evidenced by BET analysis and reduced cell wall thickness and wood density (Figure 4 ; Figures [Link] , [Link] and S7 ; Gerber et al ., 2014 ). These nano‐ and micro‐structural changes did not reduce recalcitrance in NP or made wood more amenable to subcritical water extraction (Figure 7 ), probably because of the high S‐lignin content in these plants. Improved saccharification properties in PT are of interest, but severe growth inhibition makes these lines unsuitable as biorefinery feedstocks. \n 2OGD‐ suppressed lines exhibited the highest Glc yield and significantly better Glc conversion in PT and increased nanoporosity (Figure 5 ; Figures S6 and S7 ), but the reason for this improved phenotype is unclear. The chemotype of these plants was not altered (Figure 4 ), except for an increased mGlcA content (Figure S5 ), suggesting higher xylan glucuronosylation. The SEC profile of water extracts of 2OGD plants suggested presence of short loosely bound polymers, which could be related to their improved saccharification. All 2OGD lines had nominally increased growth, with the most highly suppressed line showing the significant increase in diameter and biomass (Figure 2 ), which resulted in relatively high Glc yields per stem in PT (Figure 5d ). The ultimate test of the 2OGD line using pilot scale industrial‐like acid pretreatment showed positive features, although gains and reproducibility of the pretreatment were lower than in the laboratory (Figure 8 ). To our knowledge this is the first example of genetically modified lignocellulose of a tree species that was improved in a pilot‐scale industrial‐like biorefinery process and was more productive in the field than the WT. \n SAM‐ suppressed lines A and B had among the highest Glc yields in PT and in NP (Figure 5a,b ). These positive effects could not be explained by any of the measured parameters including the biomass pyrolyzate composition (Figure 4 ), nanoporosity (Figure S7 ) or wood anatomy parameters (Figure 3 ; Figure S2 ). An increase in mGlcA compared to WT was observed in these lines, similar to the 2OGD lines (Figure S5 ), and molecular weight profiles of water extracts were similar between 2OGD‐ and SAM‐ suppressed plants (Figure 8 ), suggesting some common features of these plants. SAM‐B also grew well, providing a rare example of transgenic modification having high Glc yield per tree in NP and in PT (Figure 5 ). However, the most suppressed SAM‐C line was dwarf (Figure 2 ), which indicates that gene dosage needs to be carefully controlled in SAM‐ suppressed plants. Traits determining saccharification Several previous studies tried to find determinants of saccharification yields based on biomass variability in natural populations (Davison et al ., 2006 ; Studer et al ., 2011 ), in different genotypes obtained by breeding (Serapiglia et al ., 2013 ), in different transgenic plant collections grown in the greenhouse (Escamez et al ., 2017 ) and in the field (Pramod et al ., 2021 ), or following controlled extraction of biomass (Chang and Holtzapple, 2020 ; DeMartini et al ., 2013 ). Here we used a collection of field‐grown WT hybrid aspen and eight transgenic lines that exhibited positive changes in different saccharification parameters (Figure 5 ), to pinpoint which of 49 analysed traits characterizing tree morphology, wood anatomy, wood chemistry and wood physical characteristics are associated with improved wood saccharification. For Glc yields in NP, we confirmed that lignin content (S, G, total lignin) is a negative factor‐limiting enzymatic hydrolysis (Davison et al ., 2006 ; Mansfield et al ., 2012 ; Min et al ., 2012 ; Serapiglia et al ., 2013 ; Studer et al ., 2011 ; Van Acker et al ., 2014 ). The analysis suggested that Glc yield in NP is negatively correlated with growth (Figure 6 ). It is possible that this correlation was driven by phenotype of C4H lines. However, a negative correlation between Glc yield in NP and growth was also observed in lines with reduced xylan acetylation, and it was explained by a higher S‐lignin content among the better‐growing transgenic lines (Pramod et al ., 2021 ). Whereas there is a link between increased body weight and lignification (Ko et al ., 2004 ), it is uncertain if such relationship universally holds beyond the sets of transgenic lines analysed in these studies. Occurrence of TW was a positive factor determining Glc yields in NP (Figure 7 ). This result supports conclusions of studies in willow, which identified TW as the main determinant of high Glc yields and Glc conversion rates (Brereton et al ., 2012 ; Foston et al ., 2011 ). This relationship is explained by a higher cellulose and lower lignin contents as well as higher nano‐porosity of TW compared to normal wood (Fagerstedt et al ., 2014 ). Glc yield in PT was dependent on totally different traits than in NP (Figure 6 ). Moreover, many traits having positive impact in NP exhibited negative influence in PT. This could be interpreted as evidence of high efficiency of acid pretreatment that decreases recalcitrance due to hemicelluloses and lignin, in particular the S‐lignin that is the major form of lignin in aspen fibres. Other studies showed a negative impact S/G ratio on lignocellulose saccharification with pretreatment in samples with similar lignin content (Mansfield et al ., 2012 ; Studer et al ., 2011 ). S/G ratio is related to wood anatomy since vessel elements are enriched in G‐lignin in hardwoods (Mellerowicz et al ., 2001 ), and it is possible that low S/G ratio corresponds to high vessel‐to‐fibre ratio, thus high microporosity and better access of enzymes to cell wall. But in our data, neither G‐lignin nor vessel frequency was significantly affecting saccharification in PT (Figure 6 ; Table S3 )."
} | 5,607 |
35411053 | PMC9001656 | pmc | 4,184 | {
"abstract": "Network models and community phylogenetic analyses are applied to assess the composition, structure, and ecological assembly mechanisms of microbial communities. Here we combine both approaches to investigate the temporal dynamics of network properties in individual samples of two activated sludge systems at different adaptation stages. At initial assembly stages, we observed microbial communities adapting to activated sludge, with an increase in network modularity and co-exclusion proportion, and a decrease in network clustering, here interpreted as a consequence of niche specialization. The selective pressure of deterministic factors at wastewater treatment plants produces this trend and maintains the structure of highly functional and specialized communities responding to seasonal environmental changes."
} | 204 |
36412737 | PMC9680249 | pmc | 4,186 | {
"abstract": "The strong adhesion on dry and wet surfaces and the durability of bioinspired hierarchical fibrillar adhesives are critical for their applications. However, the critical design for the strong adhesion normally depends on fine sub-micron structures which could be damaged during repeat usage. Here, we develop a tree frog-inspired gradient composite micropillars array (GP), which not only realizes a 2.3-times dry adhesion and a 5.6-times wet adhesion as compared to the pure polydimethylsiloxane (PDMS) micropillars array (PP), but also shows excellent durability over 200 repeating cycles of attachment/detachment and self-cleaning ability. A GP consists of stiffer tips and softer roots by incorporating gradient dispersed CaCO 3 nanoparticles in PDMS micropillar stalks. The modulus gradient along the micropillar height facilitates the contact formation and enhances the maximum stress during the detaching. The study here provides a new design strategy for robust adhesives for practical applications in the fields of robotics, electronics, medical engineering, etc.",
"conclusion": "4. Conclusions Inspired by the gradient modulus on the adhesive toe pad of tree frogs, a composite gradient micropillar array (GP) with a gradually increasing elastic modulus from the base to the tip along the micropillar height was successfully designed and constructed. The soft root of GP plays a similar role as the narrow neck structure of the desert beetle, which reduces the bending stiffness of GP and thus facilitates the contact with misaligned surfaces. The high modulus tip increases the maximum stress that is required for detaching, thus enhancing the adhesion. The slightly increased roughness on the pillar top of GP increased the hydrophobicity, which contributes the stronger adhesion in wet conditions and good self-cleaning ability. Thus, GP showed adhesion of 2.3- and 5.6-times compared to the pure PDMS micropillars array under dry and wet conditions, respectively. The results not only provide a robust material with strong dry and wet adhesion, which may find wide applications in various conditions.",
"introduction": "1. Introduction To maximize the survival in the complex, dynamic natural environments, functional gradient structures have been developed in many creatures [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. For instance, the microscale setae of the ladybird beetle Coccinella septempunctata have a gradient modulus from 7.2 GPa at the root to 1.2 MPa at the tip, endowing a high flexibility at the seta tip to enhance contact formation and a stiff stalk to maintain mechanical stability [ 6 ]. The same strategy of elastic modulus gradient has also been found in the hierarchical setae of gecko, which allows the nanoscale setal tip to form good contact with the counterpart surface, generating a strong adhesion of ~100 kPa [ 3 , 8 ]. Inspired by the modulus gradient in the setae of beetle/gecko, Hensel et al. [ 9 ] achieved similar adhesions on rough and smooth substrates by two-phase cylindrical pillars that were composed of a stiff stalk and a soft tip layer, which was prepared by sequenced casting. A smaller thickness of the top layer and a flat interface between the two phases are beneficial to the adhesion performances [ 10 ]. A gradually decreasing modulus from the pillar base to tip has also been incorporated into slanted micropillars which showed strong and anisotropic lateral friction forces [ 11 ]. Tree frogs, which can easily climb on vertical or even inverted dry/wet surfaces, have inspired the design of structured adhesives for dry and wet conditions [ 12 , 13 , 14 , 15 , 16 , 17 ]. Using a poly(acrylamide)/poly(vinyl alcohol) hydrogel to mimic the hexagonal epithelial cells in the tree frog, direct solid–solid contact has been suggested to play a major contribution to the wet adhesion [ 15 ]. Chen et al. [ 17 ] investigated the shape of epithelial cells on the toe pad of the tree frog Polypedates megacephalus and found the main shape was hexagonal. Inspired by this finding, stronger friction in the corner direction was demonstrated in slimmer polydimethylsiloxane (PDMS) hexagonal pillars. Meanwhile, Iturri et al. [ 13 ] showed higher friction forces in an elongated PDMS hexagonal pillar than regular hexagonal patterned or non-structured surfaces with/without water at the contact interface. By mimicking the densely packed and oriented hard keratin nanofibrils in tree frogs [ 18 ], Xue et al. [ 19 ] developed composite micropatterns that were composed of PDMS micropillars that were embedded with polystyrene nanopillars, showing improved adhesion and friction at the same time. Inspired by the nanoconcave top of epidermal cells on tree frogs’ toe pads, micropillar arrays with micropits [ 14 ] and nanoconcaves [ 20 ] on top have been designed and showed higher wet adhesion and friction compared to the arrays of micropillars with flat tops. Meanwhile, it has been found that the keratinized layer on the toe surface has a modulus of 5–15 MPa, but the effective elastic modulus ( E eff ) of the tissue beneath the keratinized layer continuously decreases to 4–25 kPa with the increase of depth in the toe pad [ 21 ], of which the modulus gradient is opposite as compared with the setae in geckos and beetles. The gradually softened interlayer maintains the integrity of the patterned epithelial cells, increasing the adaptability to surfaces, while the large E eff on the surface is helpful for wear resistance [ 22 ]. The incorporation of the modulus gradient that is found in tree frogs into the gecko-inspired polydimethylsiloxane (PDMS) micropillar array with T-shape tips resulted in an enhancement of adhesion of 3.6-times [ 23 ]. It has been widely demonstrated that the micro- and nanopillar arrays with T-shape tips are the best structure design to gain strong normal adhesion for various materials [ 24 , 25 ]. Surprisingly, introducing the tree frog-inspired modulus gradient can even further enhance the adhesion performance of the T-shape micropillar array [ 23 ]. However, the preparation process of T-shape tips is rather complicated and the fine overhang structure in T-shape tips is rather soft and could be easily damaged during the repeating cycles of attachment/detachment, hindering the advance of T-shape adhesives toward practical applications [ 26 ]. Therefore, it is highly needed to simplify the design of micro- and nanopillar array adhesives and develop robust adhesives with prominent adhesion abilities and durability. Here, we design a gradient composite micropillars array (termed as GP) with a modulus gradually increasing from the micropillar base to tip, mimicking the tree frog’s modulus gradient ( Figure 1 ). The GP presents 2.3-times dry adhesion and 5.6-times wet adhesion as compared to the pure PDMS micropillars array (PP) with excellent durability. The softer base in the GP allows the pillar to adapt to the contacting surface easily, forming reliable contacts. The rigid tip increases the detaching stress and, therefore, enhances the force that is required for the separation. The concept of GPs and the fabrication method can be extended to other material combinations for strong adhesions.",
"discussion": "3. Results and Discussion 3.1. Construction of GP The GP was fabricated by a soft lithography process [ 27 , 28 ], as shown in Figure 2 a, and detailed in the experimental section. Briefly, the PDMS/CaCO 3 mixture was spread onto the PU mold, and filled into the cavities by a vacuum-assisted capillary process ( Figure 2 (ai,ii)). CaCO 3 nanoparticles (NPs) were chosen as the filler based on the following reasons: (1) CaCO 3 is an abundant mineral, occupying 5% of the earth’s crust [ 29 ]; (2) the preparation of CaCO 3 NPs is simple and the size is controllable; (3) CaCO 3 NPs often serve as reinforcement to improve the mechanical strength of the polymer matrix [ 30 , 31 ]. Nearly 97% CaCO 3 NPs possess a diameter less than 1 μm, which allows them to fill into the PU mold easily ( Figure S1, Supporting Information ). The excess mixture of PDMS/CaCO 3 on the PU mold was carefully removed. Due to the larger density of CaCO 3 NPs (2.9 g/cm 3 ) than PDMS (1.1 g/cm 3 ), the CaCO 3 NPs were propelled towards the bottom of the cavities in the PU mold by applying a centrifugal force along the axial direction of the cavities ( Figure 2 (aiii)). The following coating of pure PDMS precursor on the mold and curing at 90 °C for 1 h resulted in the composite micropillars with a gradient-distributed CaCO 3 NPs, which is termed as GP in the following text ( Figure 2 a and Figure S2, Supporting Information ). For the controls, pure PDMS micropillars array (PP) and PDMS/CaCO 3 homogeneous composite micropillars array (HP) were also fabricated in the same way, but without the addition of CaCO 3 NPs or the centrifugation process, respectively. Due to simplicity of the methods, four different initial concentrations of CaCO 3 NPs in PDMS precursor ( c cal of 10, 30, 50, and 70 wt%) were used to regulate the E eff of GP and HP. For convenience, the initial c cal is used to identify the samples, such as HP 10wt% and GP 10wt% , in the following text, although the exact c cal in the pillars could be different from the initial ones. The resulting micropillars possess good physical integrity. The 3D images showed that the resulted GP, PP, and HP are 40 μm in diameter, 35 μm in height, and 80 μm in period ( Figure 2 b and Figure S3a,b, Supporting Information ). Under dark-field illumination, the backing layer in GP (HP) is black, while the micropillars in GP are shining. It suggests the presence of CaCO 3 NPs in the micropillars but not in the backing layer ( Figure 2 c). The SEM image clearly shows the presence and the gradually increased content of CaCO 3 NPs from the base to the tip in the pillars of GP ( Figure 2 d). In contrast, the homogeneous distribution of CaCO 3 NPs was found in HP ( Figure S3c,e, Supporting Information ) and no CaCO 3 NPs in PP ( Figure S3d,f, Supporting Information ). The gradient modulus of micropillars in GP was quantitatively characterized [ 32 ] ( Figure 2 e). In order to conveniently characterize the gradient, the micropillar was evenly divided into top, middle, and bottom layers along the micropillar height and the c cal of each layer was calculated based on the atom ratio of calcium to silicon. Clear gradient distributions of CaCO 3 NPs in the three layers were detected in GPs, while a uniform distribution of CaCO 3 NPs was found in HP ( Figure S4, Supporting Information ). Since the modulus of CaCO 3 is much larger than that of PDMS (26 GPa vs. 2.2 MPa), the layer with the larger c cal possesses a larger E eff ( Figure S5, Supporting Information ). Under the centrifugation at 3000 rpm for 5 min, E eff of the top layer increased to 12.0 ± 0.9 MPa, while E eff of bottom layer was 8.4 ± 1.3 MPa ( Figure 2 e). Increasing the centrifugation speed to 3900 rpm for 10 min, E eff steeply increased to 16.0 ± 0.6 MPa in the top layer and decreased to 5.9 ± 0.1 MPa in the bottom layer, forming a distinct gradient in GP 70wt% ( Figure 2 e). The increase in initial c cal , centrifugal speed and time increases the E eff of the top layer and decreases the E eff of the bottom layer. The modulus difference between the top and bottom layers divided by height is defined as the gradient rate ( Figure 2 f). For HP, the gradient rate is 0. The gradient rate of GP 70wt% reached 101.4 kPa/μm under the centrifugation at 3000 rpm for 5 min and increased to 288.5 kPa/μm under 3900 rpm for 10 min. When the initial c cal was less than 50 wt%, GP can’t reach the largest gradient rate found in GP 70wt% ( Figure S6a,b, Supporting Information ). However, the largest gradient rate of 387.7 kPa/μm was realized in the GP 50wt% under a centrifugation at 3500 rpm for 10 min ( Figure S6c, Supporting Information ). It is assumed to be the result of a moderate viscosity of the mixture. Therefore, we can precisely regulate the gradient rate of the micropillars by combining c cal with the centrifugal parameters to mimic the gradient modulus that is found in tree frog toe pads [ 21 ]. 3.2. Adhesion Performances Adhesion performances of micropillars were conducted by macroscopic and microscopic tests. Samples (6 × 6 mm) were finger-pressed onto an upside-down glass surface and weight was hung beneath ( Figure 3 a). The GP could hold the highest weight of 45 g, much higher that on PP (29 g) and HP (32 g), which clearly suggests the advantage of the gradient modulus of GP in promoting adhesion abilities. Detailed examinations on the dependence of microscopic adhesion force, F ad , and the loading force ( F L ) were carried out on a home-made device with a 5 mm glass sphere as the probe ( Figure S7, Supporting Information ) [ 23 ]. A large F L can compensate the possible misalignment between the micropillars and the spherical probe, resulting in a better contact and, therefore, a larger F ad [ 20 ]. Thus, a higher F ad was detected under a larger F L ( Figure 3 b). For instance, the F ad of PP slight increased from 0.7 ± 0.1 to 0.8 ± 0.1 mN when F L was increased from 0.5 to 5.0 mN. In contrast, the F ad of GP 214.6 (subscript indicates the gradient ratio) greatly increased from 1.1 ± 0.2 to 1.9 ± 0.1 mN, representing a 72.7% increase. The F ad of GP 214.6 under F L of 5.0 mN is 122% and 61% higher than PP and HP, respectively. The GP with c cal of 10 wt%, 30 wt% and 50 wt% all showed enhancement in F ad , but the enhancement was less than GP with c cal of 70 wt% ( Figure S8, Supporting Information ). Thus, GP 214.6 showed not only a much higher adhesion than PP and HP, but also a much stronger dependence on F L . The dependence of F ad on the gradient rate (under F L = 5 mN) was further investigated ( Figure 3 c). The F ad of HP increased from 13.9 ± 0.8 to 21.3 ± 0.4 kPa while c cal increased from 0 to 70 wt%, which indicates a positive effect of increasing E eff on the F ad . With c cal of 10 wt% and 30 wt%, the F ad slightly increased with the increase in gradient rate. At a c cal of 50 wt%, the F ad increased to 27.2 ± 1.1 kPa at gradient rate of 254.7 kPa/μm. When the initial c cal was set to 70 wt%, the F ad reached 31.9 ± 1.8 kPa at a gradient rate of 214.6 kPa/μm, which was 2.3-times the PP. The results further confirm that the enhanced adhesion originated from the modulus gradient. The further increase in the gradient rate (for instance, in GP 288.6 ), however, reduced the F ad . The reduction in F ad could be the result of the large agglomeration in the pillars ( Figure S9, Supporting Information ), which may hinder the effective transfer of stress during the detachment. Furthermore, increasing the aspect ratio (AR) of micropillars could enhance the compliant ability of the micropillars to the counterpart surface and, therefore, the adhesion ( Figure 3 d) [ 33 ]. Therefore, the F ad of GP 241.6 was 46.9% improved when AR was increased from 0.88 to 1.50 with the same c cal and centrifugal conditions. Interestingly, the adhesion enhancement in GP was also stronger than that of HP (28.1%) and PP (18.2%). As GP has no submicron structures, such as the overhangs in T-shape micropillars, GP 241.6 has no notable decay in F ad after 200 macroscopic cycles test ( Figure 3 e), suggesting an extraordinary durability of the GP adhesive. Macroscopic adhesion on a wet surface was also investigated ( Figure S10, Supporting Information ). As compared to the adhesion on the dry surface, wet F ad of GP was much smaller. A wet F ad of 5.8 ± 1.0 kPa was detected on GP 214.6 with deionized water at the contacting interface ( Figure 3 f). It is reasonable as the captured liquid at the contacting interface hinders the effective formation of contact, reducing the adhesion. On the other hand, however, the wet F ad of GP 214.6 remained the best as compared with PP and HP, which was 5.6- and 2.1-times the PP (1.0 ± 0.1 kPa) and HP (2.8 ± 0.4 kPa), respectively. Once again, it demonstrated the merits of the incorporation of modulus gradient in a micropillar array for adhesion enhancement, in both dry and wet conditions. The gradient modulus in GP contributes to the adhesion enhancement based on two mechanisms. Gorb et al. [ 34 ] found a narrow neck beneath the contacting tip of the seta of the desert beetle Dytiscus marginalis and suggested that the narrow neck could reduce the local bending stiffness, making it easy to adapt to the misaligned surfaces. The soft root in GP could possess a similar function as the narrow neck. To demonstrate the concept, the approaching of a surface with tilting angle of 3° to the GP tip was finite element simulated ( Figure 4 a). When approached to a tilted surface, the micropillar bends to facilitate the contact formation [ 23 ]. While PP has the smallest E eff and is the easiest to bend, it needs 4.8 mN to form full contact with the tilted surface ( Figure 4 b). In contrast, HP needs a much larger F L of 11.5 mN to form full contact. GP 370 needs a F L of 5.4 mN to form the full contact, which is quite close to the F L that is required for PP. On the other hand, GP with a much larger gradient rate of 85 kPa/μm (GP 85 ) could form full contact under a F L of 10.9 mN, which is similar to HP. In contrast, although GP also possess a large E eff at the free end similar to HP, the soft root of GP increases its flexibility, enhancing the attaching ability to uneven or misaligned surfaces. The larger the gradient ratio, the smaller F L is needed for GP to adapt to the tilted surface ( Figure 4 b). GP generates a larger stress maximum at the contact interface ( σ ), which is determined by the work of adhesion ( W ) and the effective modulus of the pillar tip ( E eff-tip ) [ 35 , 36 ]: σ = W E eff − tip A \nwhere A is the area of the pillar end. As the detaching pairs are identical in all the cases here, W and A are constants. Therefore, the increase of E eff-tip would increase the σ ( Figure 4 c), which means a larger force ( F ad ) is needed to separate the contacting pair. For instance, GP with E eff-tip of 15 MPa has a σ of 50 kPa, which is almost 2-times of that in PP and HP. The easier contact formation and the larger stress that is required for detachment, therefore, generate the strongest adhesion on GP as compared with HP and PP. 3.3. Applications of GP Undesirable damage occurring on the contact surfaces is a common phenomenon when grasping soft objects. In order to avoid this kind of damage, superior adhesion is in great demand on grasping soft objects [ 37 ]. Attempts to improve the adhesion performance on soft surfaces have mainly been pursued by the use of special adhesives or octopus-inspired sucker structures [ 12 , 37 ]. Since the soft objects are vulnerable, any excessive external force should be avoided. Therefore, it is highly desirable to develop a reusable adhesive pad with extraordinary adhesion ability with little external force. The strong adhesion of GP can solve this challenge perfectly. A soft plasticine toy with surface roughness (Sa) of 3.91 μm was used to demonstrate the concept ( Figure S13, Supporting Information ). The excellent adhesion of GP allows it stick to the surface of the soft plasticine toy by applying an ignorable F L . The higher adhesion under small F L makes GP capable of picking up an object with smaller F L as compared to PP and HP ( Figure 3 b). The subsequent peeling at a small angle can release the toy to a designated position easily ( Figure 5 a). The surface showed no clear deformation as compared to the surface before transportation ( Figure 5 b). In contrast, although a small force was carefully applied, sharp pliers grasping strongly deformed the surface, leaving bite marks on the surface ( Figure 5 c,d). After transferred by GP five times, the surface roughness of the soft plasticine toy slightly increased to 7.67 μm ( Figure 5 e). However, the surface roughness increased significantly to 59.88 μm after the transferring with the sharp pliers grasping five times. 3.4. Self-Cleaning Ability of GP The self-cleaning ability is important for the re-usage of GP in dirty environments. The geometry of the micropillar array and the larger roughness on the pillar end of GP (Sa = 28.2 ± 4.9 nm, Figure S12 ) offer GP a water contact angle of 147.9 ± 0.9°, which is slightly larger than HP and PP ( Figure 6 a). The good hydrophobicity endows GP with self-cleaning ability [ 38 ]. With a simple flushing with water flow, GP can fully recover its adhesion ability after contamination by dust ( Figure 6 b). After 10 cycles of soiling and cleaning, the adhesion of GP remained unchanged ( Figure 6 c). It thus undoubtedly demonstrates the robustness of GP in dirty conditions and the ability of self-cleaning, which are of great significance to the reusability application of GP in real environments."
} | 5,249 |
26296065 | PMC4817675 | pmc | 4,188 | {
"abstract": "Bacteria play a central role in the cycling of carbon, yet our understanding of the relationship between the taxonomic composition and the degradation of dissolved organic matter (DOM) is still poor. In this experimental study, we were able to demonstrate a direct link between community composition and ecosystem functioning in that differently structured aquatic bacterial communities differed in their degradation of terrestrially derived DOM. Although the same amount of carbon was processed, both the temporal pattern of degradation and the compounds degraded differed among communities. We, moreover, uncovered that low-molecular-weight carbon was available to all communities for utilisation, whereas the ability to degrade carbon of greater molecular weight was a trait less widely distributed. Finally, whereas the degradation of either low- or high-molecular-weight carbon was not restricted to a single phylogenetic clade, our results illustrate that bacterial taxa of similar phylogenetic classification differed substantially in their association with the degradation of DOM compounds. Applying techniques that capture the diversity and complexity of both bacterial communities and DOM, our study provides new insight into how the structure of bacterial communities may affect processes of biogeochemical significance.",
"introduction": "Introduction Carbon (C) cycling has received considerable attention in recent years, spurred by the increase of carbon dioxide concentrations in the atmosphere and the therewith-associated changes in climate ( Solomon et al. , 2007 ). In the wake thereof, attempts have been made to balance the global C budget and to develop a mechanistic understanding of its underlying dynamics. This has led to a revision of the traditional view in which inland waters were considered a passive ‘pipe' that merely transported C from land to sea. It is now, however, recognised that inland waters make up an active compartment: one that mineralises, transforms and stores C of terrestrial origin besides transporting it to the oceans ( Cole et al. , 2007 ; Battin et al. , 2009 ; Tranvik et al. , 2009 ). Therefore and in view of future climatic changes, it is of great importance to comprehend which factors influence the mineralisation and transformation of terrestrially derived C in freshwater ecosystems. It is the bacteria that essentially decompose this allochthonous dissolved organic matter (DOM) and introduce it into the aquatic food web ( Pomeroy 1974 ; Azam et al. , 1983 ; Jansson et al. , 2007 ). Bacterial degradation of DOM is carried out by phylogenetically diverse communities, whose composition has been shown to be affected by the quality and quantity of DOM (for example, Logue and Lindström, 2008 ). Furthermore, differences in bulk bacterial processes (for example, bacterial respiration or production) related to changes in DOM quality and quantity point towards the existence of functionally distinct bacterial groups (for example, Kirchman et al. , 2004 ). Yet, studies investigating how the composition of bacterial communities affects the cycling of C in fresh waters have to date yielded inconclusive results; while some argue to having observed a close relationship between bacterial community composition (BCC) and C processing ( Crump et al. , 2003 ; Kirchman et al. , 2004 ; Judd et al. , 2006 ; Kritzberg et al. , 2006 ; Langenheder et al. , 2006 ; Bertilsson et al. , 2007 ), others found inconsistent ( Comte and del Giorgio, 2009 , 2010 ; Lindström et al. , 2010 ) or weak links ( Langenheder et al. , 2005 ). It has to be noted, though that rather than actually demonstrating a direct relationship between BCC and C processing (see Langenheder et al. , 2005 , 2006 ), most studies illustrate that environmental parameters, such as DOM quality and quantity, affect community composition and functioning alike. Given the intertwined nature of BCC, the environment and bacterial functioning, studies directly addressing the relationship between aquatic BCC and C processing are clearly lacking. This lack may be partly due to former methodological limitations. Despite their importance in aquatic systems, DOM and microbial diversity yet remain to be characterised for the most part ( Curtis and Sloan, 2005 ; Hertkorn et al. , 2008 ). As DOM is one of the most complex molecular mixtures on Earth ( Hedges et al. , 2000 ) and microbial communities are extremely diverse ( Curtis and Sloan, 2004 ), studies going beyond bulk assessments of DOM as well as the most abundant members of microbial communities have been rather challenging. Recent technological advances in the field of molecular biology (for example, high-throughput sequencing) and adopting advanced instrumental approaches into analytical chemistry (for example, electrospray ionisation mass spectrometry (ESI-MS)) have, however, made it possible to obtain information of greater resolution and depth in this respect (see Kujawinski, 2011 for an overview and Herlemann et al. , 2014 ; Landa et al. , 2014 and Shabarova et al. , 2014 for studies that combine the two approaches). Such an in-depth and integrative characterisation of both complex DOM compounds and microbial communities is a prerequisite for exploring the relationship between microbial community composition and the processing of DOM. Here we studied the link between the composition of aquatic bacterial communities and the degradation of DOM of terrestrial origin. The aim was to examine how bacterial communities different in composition differ in their processing of DOM. We hypothesised that bacterial assemblages of different origin differ in their ability and potential to degrade DOM, because they vary in composition. We tested this hypothesis by adopting a common garden experiment in which a uniform, terrestrially derived yet artificially prepared DOM medium was inoculated with aquatic bacterial communities collected from four sites of varying environmental character.",
"discussion": "Discussion Taking advantage of technological advances in analytical chemistry and molecular biology, we explored how the composition of aquatic bacterial communities affected the degradation of DOM of terrestrial origin. Having adopted an experimental approach in which model communities were exposed to a terrestrially derived DOM substrate, our results highlight that although bacterial communities that differ in composition degraded the same amount of DOM, both the temporal pattern of degradation and, most importantly, the compounds that were degraded significantly differed. Finally, we observed that the most abundant bacterial taxa differed substantially in their association with the degradation of DOM compounds. A major goal in ecology is to link the composition of biological communities with processes occurring in an ecosystem. Given the entwined nature of microbial community composition, the environment and ecosystem processes, one of the greatest challenges is to test for direct effects of composition on functioning. Common garden experiments allow for precisely that by standardising environmental parameters and, therefore, enabling the teasing apart of the effects of the environment from the composition of microbial communities on functioning ( Reed and Martiny, 2007 ). The downside of incubating microbial communities under batch growth conditions, however, is that the resulting community will differ from the composition of its original inoculum (for example, Christian and Capone, 2002 ). Indeed, our analyses identified a change from environmental to experimental bacterial communities in both diversity and composition ( Supplementary Figures S2 and S4 , respectively). It has been further suggested that such experiments favour micro-organisms rare in nature but featuring opportunistic, copiotrophic qualities that allow for a more rapid adaptation to changes in environmental conditions and, hence, to outcompete others that are originally more abundant. In nature, DOM varies in quality (and quantity) over space and time (for example, Kothawala et al. , 2014 ), variations to which microbes need to adapt. Yet, the exposure of different bacterial communities in our experiment to a freshly prepared, terrestrially derived and, hence, highly bioavailable DOM substrate as C source possibly enhanced growth of such naturally rare bacteria. As the communities still differ in composition at the end of the experiment, it can, however, be assumed that the functional differences observed are the consequence of initial compositional differences among the bacterial communities. Our results, thus, show a close link between BCC and function. Going beyond a mere identification of a link between BCC and DOM degradation, our results further highlight that the four experimental communities degraded different components of the DOM pool. Although fluorescence analyses illustrate that certain DOM components were commonly more bioavailable than others, both fluorescence and ESI-MS analyses demonstrate that the four different bacterial communities differed in which DOM components were degraded preferentially. Most importantly, ESI-MS analysis uncovered that community composition was of little importance regarding the degradation of LMWC, whereas the utilisation of masses of greater size differed among communities. This means that the ability to use LMWC is a functional property (trait) rather common in all of the four bacterial communities, whereas the capability to use C of high-molecular-weight appears to be a trait restricted to particular bacterial communities. An explanation could lie in a finding made by Weiss et al. (1991) that compounds of up to ~600 Da (that is, LMWC) can be taken up readily by micro-organisms across the cell membrane (that is, through a variety of transmembranic transport systems), whereas larger ones require extracellular cleavage by means of enzymatic hydrolysis (that is, via individual or interacting ectoenzymes), an ability that indeed not all bacterial taxa possess (for example, Berlemont and Martiny 2013 ). Yet, bacterial members within a community vary not only with regard to the ability to produce ectoenzymes but also in their capability to express transmembranic transport systems that allow the uptake of compounds exceeding 600 Da (for example, Teeling et al. , 2012 ). However, a microbial community's toolbox of traits is more than the sum of its parts; on the one hand, some bacterial taxa may be needed to actually facilitate the degradation process, allowing other micro-organisms to either hydrolyse substrates further or take them up, on the other the process may only continue when some microbes act in concert. Pedler et al. (2014) , for instance, demonstrated that the readily available fraction of a coastal DOM pool could be completely removed by a single taxon, whereas decomposition of the less bioavailable portion required additional members of the community. Hence, it becomes apparent that not only the chemical composition of DOM (that is, quality) but also the distribution of traits within microbial communities are important when it comes to whether or not DOM evades microbial re-mineralisation and transformation. As such, bioavailability can be perceived as an ongoing interaction between the chemical composition of DOM and a microbial community's metabolic capacity rather than merely an inherent property of DOM ( Nelson and Wear 2014 ). Although the degradation of either low- or high-molecular-weight carbon was not restricted to a single phylogenetic clade, our results illustrate that bacterial taxa of similar phylogenetic classification differed substantially in their association with the degradation of DOM compounds (both at a 97% and 99% sequence identity level; results for the latter are not shown). This may be an indication for high variation in the functional, and thus ecological, potential among closely related populations within microbial communities (that is, micro-diversity; see Zimmerman et al. , 2013 ); for example, the two as Herminiimonas classified bacterial taxa C3 and C836 were generally associated with the degradation of low- and high-molecular-weight carbon, respectively. Hence, our results demonstrate that the capacity of a community to degrade DOM compounds cannot easily be predicted from phylogenetic information alone, at least not from information derived from the 16S rRNA gene (see also Covert and Moran, 2001 ; Fuhrman and Hagström, 2008 and Martiny et al. , 2013 ). Considering the associations observed between the relative abundances of the most abundant bacteria and the degradation of DOM compounds the question arises ‘Why do the observed degradation patterns not look more similar, given that these bacterial taxa were generally present in all four communities?'. One explanation could be that the functional gene repertoire of these bacteria varied between experimental communities as a result of adaptation to their original environments. Another could be that these abundant bacteria depend on other taxa with a different set of traits fundamental to the degradation of certain DOM compounds (see Pedler et al. , 2014 ); taxa that are rarer and may not be present in all communities. Such interplay will, though, not be detectable via correlation analysis. In fact, caution has to be exercised when interpreting the results from the correlation analysis in that it does not allow drawing conclusions about the cause and effect, and, as such, cannot be used to unambiguously link a specific bacterial taxon to the degradation and utilisation of a particular DOM compound. In addition, size (for example, m/z ) represents only one property of DOM; correlating other properties with bacterial taxa may yield more nuanced and different associations, as well as trait-specific insights. Once associations have been established, they may guide researchers to conduct studies more non-generic in character, such as controlled experiments in which the degradation capacities of a single bacterial population are investigated. Moreover, identifying functional genes involved in the degradation of DOM along with assigning the chemical composition to individual DOM compounds via ultrahigh-resolution MS (for example, Fourier transform ion cyclotron resonance MS; see Hertkorn et al. , 2008 ) could potentially provide insight into microbial traits that may or may not be phylogenetically constrained. Combining such trait-based information with knowledge of the regulation of microbial activities, the monitoring of functional genes (metatranscriptomics or metaproteomics; for example, Moran, 2009 ; Teeling et al. , 2012 , respectively) and/or metabolic features (single cell genomics; for example, Rinke et al. , 2013 ) may offer a way to explore the use of individual organic matter compounds by specific microbial taxa in complex communities to an even greater depth and improve our understanding of how microbial community composition may affect the cycling of C in the biosphere."
} | 3,771 |
35399376 | PMC8983376 | pmc | 4,189 | {
"abstract": "Bioaugmentation, the addition of cultured microorganisms to enhance the currently existing microbial community, is an option to remediate contaminated areas. Several studies reported the success of the bioaugmentation method in treating heavy metal contaminated soil, but concerns related to the applicability of this method in real-scale application were raised. A comprehensive analysis of the mechanisms of heavy metal treatment by microbes (especially bacteria) and the concerns related to the possible application in the real scale were juxtaposed to show the weakness of the claim. This review proposes the use of bioaugmentation-assisted phytoremediation in treating heavy metal contaminated soil. The performance of bioaugmentation-assisted phytoremediation in treating heavy metal contaminated soil as well as the mechanisms of removal and interactions between plants and microbes are also discussed in detail. Bioaugmentation-assisted phytoremediation shows greater efficiencies and performs complete metal removal from soil compared with only bioaugmentation. Research related to selection of hyperaccumulator species, potential microbial species, analysis of interaction mechanisms, and potential usage of treating plant biomass after treatment are suggested as future research directions to enhance this currently proposed topic.",
"conclusion": "6 Conclusions Bioaugmentation is not suitable to be applied alone in treating heavy metal contaminated soil in real-scale area due to incomplete separation of heavy metals (and its intermediate compounds) from the treated medium after the treatment. Several research works report successful heavy metal removal from contaminated soil that are mostly conducted at the laboratory scale under a controlled environment. The said research studies also limitedly discuss the separation of heavy metals from the medium to obtain pollutant-free soil after treatment. In addition, separation technology used in laboratory scale is considered applicable for use in real application due to extensive energy consumption and difficult operation. Phytoremediation is suggested for use as heavy metal contaminated soil treatment method, whereas bioaugmentation of PGPB to assist the phytoremediation is proven to be the future promising technology with higher removal efficiencies. Searching for potential hyper accumulator plant species of certain heavy metal type, potential microbial species, and its interaction with plants during bioaugmentation-assisted phytoremediation, fate of heavy metals, and post treatment handling of produced plant biomass are future research directions to be explored further to enrich the knowledge of the treatment of heavy metal contaminated soil by bioaugmentation-assisted phytoremediation.",
"introduction": "1 Introduction Heavy metal is commonly found on Earth and is widely acknowledged as metalloids and metals in the periodic table ( Yadav et al., 2019 ). The property of heavy metal with its high atomic weight and density makes it a good candidate for conducting electricity ( Jaishankar et al., 2014 ; Shadman et al., 2019 ). Heavy metal processing industries are increasing with the utilization of heavy metals in daily life ( C. He et al., 2020 ). The increment of heavy metal utilization leads to increasing cases of heavy metal pollution that is very dangerous to the environment and human life ( Hejna et al., 2018 ). Several heavy metals such as chromium in hexavalent form, Cr(VI), lead (Pb), and arsenic, which is generated from anthropogenic activities, are poisonous to living organisms ( Oliveira, 2012 ; Kamaruzzaman et al., 2019 ; Titah et al., 2018 ). Mercury (Hg) also poses several hazardous characteristics such as neurotoxicity and immunotoxicity to living organisms, including humans ( Bjørklund et al., 2017 ). Arsenic also causes enzyme reaction inhibition, especially related to phosphate uptake and utilization ( Titah et al., 2018 ). Cadmium (Cd), which is abundantly found in soil, could reduce soil fertility due to its hazardous properties ( Li et al., 2019 ). Specifically, Cd in soil is directly transferred to most plants and animals, inhibiting their actual growth and function ( Chibuike and Obiora, 2014 ; Scaccabarozzi et al., 2020 ). Humans may also be affected and may possess health risks such as lung cancer and kidney problem because food sources are contaminated with toxic heavy metals ( Li et al., 2019 ). Cd and Cr mining could also cause severe environmental pollution and an ecological disaster as it involves large operating areas. Mining activities, as one of the most reported sources of heavy metal in soil, release heavy metals into the surrounding environment ( Ighalo et al., 2022 ). They cause most organisms to become exposed to hazardous pollutants and lose their natural habitat, hence creating another alarming issue of resistant bacteria in the environment ( Oladipo et al., 2018 ; Schippers et al., 2010 ). The abundance of toxic waste accumulated in the environment has a remarkable effect on the entire ecosystem ( Igwegbe et al., 2022 ). Therefore, the effect of anthropogenic activities should be reduced to minimize heavy metal pollutants in nature. Bioaugmentation is a widely known approach to remediate heavy metal from contaminated environment by adding indigenous and exogenous microorganisms that can resist and reduce the toxicity of heavy metals ( Hassan et al., 2019 ; Purwanti et al., 2020 ). Indigenous microorganisms are isolated from contaminated soil and reinoculated back into the corresponding contaminated soil ( Purwanti et al., 2018a , Purwanti et al., 2018b ). By contrast, exogenous microorganisms are isolated outside the contaminated areas and introduced into the desired contaminated area ( Huang and Ye 2019 ). Several of the processes involved in remediating heavy metals using microbes are biosorption, bioaccumulation, bio chelation, bio digestion, biomineralization, and biotransformation ( Nwaehiri et al., 2020 ). Several studies claimed the success of bioaugmentation in treating heavy metal contaminated soil. Mahbub et al. (2017) reported the successful removal of Hg from artificial contaminated soil using Sphingobium SA2 with removal efficiency reaching 50%. Ibarrolaza et al. (2011) also mentioned the removal of hexavalent chromium from artificial contaminated soil by Sphingomonas paucimobilis 20006FA with removal efficiency of 90%. Polti et al. (2014) also reported removal of hexavalent chromium from artificial contaminated soil by using consortium of Streptomyces sp. M7, Streptomyces sp. MC1, Streptomyces sp. A5, and Amycolatopsis tucumanensis with removal efficiency of 86%. However, most of the treatments were conducted in laboratory scale using artificial contaminated soil under controlled condition. Questions related to the applicability of this method in real-scale contaminated soil, especially in terms of the segregation of deposited metal from soil, to obtain remediated clean medium free from toxic heavy metals were elevated ( Agnello et al., 2016 ; Purwanti et al., 2019a ). Even if toxic metals are deposited as stable complexed precipitates in soil, stable heavy metals may turn back into mobilized phase due to uncontrolled climate or weather changes. This review highlights several concerns related to the applicability of bioaugmentation in treating contaminated soil, focusing on the failure of separating soil with metal after treatment. This review article identifies the mechanism of bacteria in detoxifying heavy metal correlated with the removal mechanisms occurring during the bioaugmentation in treating heavy metal contaminated soil. Phytoremediation is suggested as an alternative solution for the concerns, whereas the introduction of plant growth promoting rhizobacteria inside the heavy metal contaminated soil phytoremediation system will alleviate the removal processes. This review is expected to shed a light on the low-real-scale applicability of bioaugmentation to treat heavy metal contaminated soil while also providing the application of phytoremediation as a promising approach to treat heavy metal contaminated soil."
} | 2,037 |
36738824 | PMC10020420 | pmc | 4,190 | {
"abstract": "Understanding why animals organize in collective states is a central question of current research in, e.g., biology, physics, and psychology. More than 50 years ago, W.D. Hamilton postulated that the formation of animal herds may simply result from the individual‘s selfish motivation to minimize their predation risk. The latter is quantified by the domain of danger (DOD) which is given by the Voronoi area around each individual. In fact, simulations show that individuals aiming to reduce their DODs form compact groups similar to what is observed in many living systems. However, despite the apparent simplicity of this problem, it is not clear what motional strategy is required to find an optimal solution. Here, we use the framework of Multi Agent Reinforcement Learning (MARL) which gives the unbiased and optimal strategy of individuals to solve the selfish herd problem. We demonstrate that the motivation of individuals to reduce their predation risk naturally leads to pronounced collective behaviors including the formation of cohesive swirls. We reveal a previously unexplored rather complex intra-group motion which eventually leads to a evenly shared predation risk amongst selfish individuals.",
"conclusion": "5 Conclusion In summary, we have investigated the intra-herd dynamics which evolves within a group of individuals competing with each other to reduce their predation risk. Using a MARL framework, we demonstrated that group members first assemble in a cohesive group which exhibits pronounced collective properties. Depending on the specific reward parameters, groups with different topologies are observed, ranging from strongly to weakly rotating groups and rings. Notably, the selfish motivation of individuals to minimize their DOD eventually leads to a fair risk distribution amongst all group members. Our results therefore indicate that Hamilton’s hypothesis indeed provides an explanation to describe gregarious behavior in living systems.",
"introduction": "1 Introduction Many animal species organize in groups, e.g., to gain benefits regarding foraging efficiency ( Sumpter, 2010 ), temperature control ( Szopek et al., 2013 ), energy considerations ( Trenchard and Perc, 2016 ) or predation risk ( Turner and Pitcher, 1986 ). The formation of such collective states is often described by social interaction rules which indeed leads to the formation of flocks, swirls and swarms as observed in many living systems ( Galton, 1871 , Couzin et al., 2003 , Jens Krause, 2002 , Parrish et al., 2002 ). As an alternative approach, W.D. Hamilton postulated the selfish herd hypothesis (SHH) which suggests that group formation of prey results from their selfish motivation to lower the predation risk ( Hamilton, 1971 ). Within Hamilton’s simplified two-dimensional model, the predation risk of each individual is proportional to the area A of its Voronoi polygon ( Boots et al., 1999 ), the latter being typically referred to as the domain of danger (DOD). When each individual attempts to minimize its DOD, this naturally leads to the formation of dense groups. Evidence supporting such geometric measure of predation risk is obtained from e.g., fiddler crabs ( Viscido et al., 2002 ), seals ( De Vos and O’Riain, 2010 ) and birds ( Quinn and Cresswell, 2006 ). Although Hamiltons concept looks straightforward, the necessary motional strategies of individuals can be rather complex due to their competition for small DODs. In previous agent-based numerical simulations, the SHH has been studied by using simplifying motional rules aiming to achieve minimization of DODs ( Hamilton, 1971 , Viscido et al., 2002 , Morton et al., 1994 , James et al., 2004 , Ose and Ohmann, 2017 , Algar et al., 2019 ). However, such approaches do not warrant an optimal strategy of individuals. Furthermore, it typically leads to freezing of the group once agents have reached a minimal distance to neighbors. Therefore previous studies have mainly studied the transient process of group formation ( Morrell et al., 2011 ) but did not investigate the complex intra-herd dynamics after aggregation. In our study, we are using Reinforcement Learning (RL) to identify the unbiased and optimal strategy of selfish agents competing for their DODs. Compared to previous studies, this approach avoids the assumption of motional rules or social forces (alignment or cohesion) but provides an unbiased solution to the SHH. RL has already proven to be a useful tool for replicating natural behaviors, e.g., in the context of optimal response to predators and food sources ( Durve et al., 2020 , Young and Manh La, 2020 , López-Incera et al., 2020 ). In our study we demonstrate the application of RL in the context of Hamiltons hypothesis. We observe the formation of a cohesive group which surprisingly displays a global rotation which has not been found in previous SHH-related simulations. In addition, we observe that agents permanently travel between the center and the edge of the group which eventually leads to an identical time-averaged DOD of all group members. This suggests that the selfish motivation of agents to reduce their DODs eventually leads to an equal predation risk of all group members.",
"discussion": "4 Discussion Using MARL we trained a group of selfish individuals to minimize their predation risk by reducing their DODs. In contrast to most previous SHH studies that relied on specific motional rules, in our approach we let the behavior emerge as an unbiased solution to the task. In agreement with Hamilton’s hypothesis ( Hamilton, 1971 ), we found that individuals gather into cohesive groups. Notably, these groups exhibit a complex intra-herd dynamics leading to group rotation as observed in many living systems ( Delcourt et al., 2016 ). Opposed to numerical simulations where rotating states are usually obtained by imposing alignment interactions between agents ( Couzin et al., 2002 , Vicsek et al., 1995 ), here such behavior results from the attempt of the agents to minimize their DODs. The appearance of swirls in context of the SHH can be understood by considering that edge positions of the group are less favorable (due to their larger DODs) compared to those near the group center. As a result, the motion of agents at the edges towards the groups center is rewarded. In addition, however, individuals aim to avoid collisions since close encounters are penalized. Keeping a balance between these two effects, eventually results in a preferred distance to neighbors which generally leads to group rotation ( Delcourt et al., 2016 , Vollmer et al., 2006 , Nuzhin et al., 2021 ). Most strikingly, we observe a fair risk distribution established amongst selfish individuals. This seems to be surprising because one would expect that agents near the group center would never abandon such positions having low (compared to edge positions) predation risk. However, agents do not have full control regarding their DODs because they also depend on the positions of their neighbors. As a result, agents must permanently respond to the motion of their peers which causes the system to remain fully ergodic. Therefore all agents will move through entire group which then leads to identical time-averaged DODs. We wish to emphasize that all behaviors reported in our study remain stable upon variations of parameters such as the vision angle, the strength of noise and even when visual blocking of neighbors is entirely removed. In all cases, an equal distribution of risk is established over time."
} | 1,874 |
34283628 | null | s2 | 4,191 | {
"abstract": "We report that "
} | 3 |
34841332 | PMC8610361 | pmc | 4,192 | {
"abstract": "Highlights • Electron transfer mechanism elaborated for various fungal species. • Application of fungi as a cathode and anode catalyst is discussed. • Different factors affecting on performance of fungi based MES are reviewed. • Fungi are classified based on their electron transfer mechanism.",
"conclusion": "15 Conclusion Fungi based MFCs are a recent advancement in wastewater treatment technology. Electrodes and microorganisms are the key players in MFCs, whose improvement and modification is directly related to the increase of the output of the system. The present review provides evidence that fungi can be explored and measured as capable microorganisms for biological fuel cells. Some yeast species can efficiently digest complex organic substances with excellent power output even at high working temperatures making them better alternatives for waste conversion. Furthermore, adding graphene to the surface of the carbon material can increase efficacy and may be commercialized in the near future because of its carbon neutral characteristics. The use of potent fungal strains as anodes and cathodes (biocathodes) is also summarized in this review. Electron transfer system in the fungal cell and various types of fungi with their electron transport mechanisms is also highlighted for the benefit of stakeholders. S. cerevisiae , with modifications in the electrode and optimization of the environmental condition have shown promising results for MFCs. Various experiments have also been summarized to prove the efficiency of fungi in the degradation of organic matter in wastewater and the simultaneous generation of bioelectricity. However, to improve the activity of fungi based MFCs, extensive research is required to design the particular alterations in electrode material and to find and improve the electron transfer mechanism of different fungal strains that are of use in energy generation. This review traces the chronological development of microbial fuel cell technology with fungi as catalysts and the different operational factors in optimizing this technology to enhance overall power production. The review also provides measures to overcome the limitations associated with this technology by developing fungal mediated MFCs including various potent strains and utilization of diverse nanomaterials for enhanced electricity generation. Nevertheless, the estimated cost of fungal mediated MFCs and cell life cycles need to be addressed more precisely in comparison to conventional methods in order to obtain more valuable information on the efficiency of this technology.",
"introduction": "1 Introduction The overwhelming demand for energy generated by the exponential increase in global population and rapid industrial growth has led to an excessive consumption of fossil fuels that has drastically depleted these resources. Generating energy from organic material/biomass is a sustainable alternative approach to address this crisis. The traditional methods of bioenergy production have certain limitations such as the need for large spaces, high capital investment and complexities associated with the production process. Several studies have reported the potential of microorganisms to reduce the cost of the bioconversion process and help to generate bioelectricity from biodegradable waste ( Ban et al., 2001 ; Narita et al., 2006 ). Research work on bioenergy generation and sustainability has, thus, increased throughout the world to address alternative, non-chemical approaches for power generation. Biotic means of bioenergy production usually exploits different types of microbial species like algae and bacteria. However, very little information is available on the use of fungal-mediated electrochemical system for energy generation. since certain fungi have only been reported for their potential to use their novel cell factories for energy generation. Considering their rapid growth and metabolism, their ability to degrade and convert waste materials for the production of bioenergy within a short period, and the presence of complex enzymatic systems in them, the prospect of using fungi in bioremediation is quite promising. For instance, microbial electrochemical technology (MET) using fungi to generate energy using wastewater as a substrate. This has a great deal of potential in future for the development of alternative renewable energy sources like utilization of oleaginous microorganisms for biodiesel production and product formulation. Biodiesel is a renewable source of energy that is of particular interest to researchers in the field of biofuel production. Biofuel can be obtained from various plant biomass, such as cereals, sugar crops and edible oil seeds, etc. Various categories of fungi have recently been reported for the production of biofuels from such biomass. Fungal strains such as lignolytic and hydrolytic are commonly being utilized for the production of bioethanol. Certain types of basidiomycetes fungi have also been reported for their potential to produce extracellular enzymes that degrade the lignocellulosic materials. The use of oleaginous microorganisms like fungi has many advantages such as reduced land requirement, short cultivation period and their production of high lipid / fatty acids like plant or vegetable oils ( Beopoulos and Nicaud, 2012 ; Ratledge et al., 2004 ). Fungal cells can be used for environmental management and bioelectricity generation since they produce certain enzymes such as peroxidases (lignin peroxidase and manganese-dependent peroxidase) that degrade hemicellulose, lignin and polyaromatic phenols ( Hofrichter, 2002 ). In recent years, yeast and filamentous fungi have been identified as oleaginous microbes of significant importance. Oleaginous fungal species within Zygomycetes are an excellent source of oleic and palmitic acids that may be used for biodiesel production. Furthermore, anaerobic fungi have an arsenal of extracellular multienzyme complexes that aid in the digestion of various biomasses for biogas production. Recent studies have highlighted the efficacy of fungal-based MFCs in biodiesel production. These potential strains can transfer electrons via cytochrome C. The Candida sp., Saccharomyces cerevisiae, Colletotrichum sp., Alternaria sp., Penicillium sp., Rhizopus sp. and Aspergillu s sp. which have been identified for their prominent role in MFC for electricity generation (Belniak and Maminska, 2018). Fungi become oleaginous when various organic substrates like glucose and sucrose are added to their growth medium. Each oleaginous microorganism has specific abilities to utilize organic substrates and enhancing the lipid yield by maximizing the bioconversion of organic substrates. It has been observed that, in a microbial consortium, a less productive strain always tends to follow a more productive strain during the co-metabolisim, resulting in the production of a higher total biomass than those of single cultures. To extract lipids, the entire fungal biomass may be utilized which has an added advantage. This microbial lipid can be directly converted to fatty acid methyl esters (FAME) using low-cost methods as mentioned in Bautista et al. (2012) . Catalysts, on the other hand, are required in large-scale biodiesel production facilities in order to accelerate the process. The lower-chain carbon compounds produced during the enzymatic treatment to wastewater can be utilized as substrate for microbial oxidation on MES. Biofuel production involves the production of biohydrogen, biodiesel and bioethanol. The zygomycetes fungi biomass, such as Mortierella isabellina and Cunninghamella echinulate have been been reported to contain 60–70% lipid content and 40–57% of dry cell weight respectively ( Fakas et al., 2009 ). Certain fungal genera, such as Aspergillus and Mucor have recently been recognised for their ability to store oils in their cells under specific conditions, with a maximum oil content of up to 80% (Dhanasekaran et al., 2017). Fungal strains with high lipid content are usually preferred, for their greater efficiency in the production of biofuels and the metabolism of triacylglycerides(TAG). Kurosawa et al. (2013) isolated a few oleaginous microorganisms that have the ability to metabolize xylose and thereby assist in lipid production from lignocellulosic hydrolysates. Yeasts are eukaryotic fungi that have a wide range of economic and environmental application. For example, Candida melibiosica, Blastobotrys adeninivorans, Kluyveromyces marxianus, Pichia polymorpha, P. anomala and Saccharomyces cerevisiae have been examined as biocatalysts in MFCs with or without an external mediator. Yeast, as a eukaryote, has gained the significant interest of researchers mainly due to its ease of use in MFCs. Kluyveromyces marxianus, one of the most promising yeast strains, produces high power output under relatively high temperature conditions when grown in natural organic substrates . The fungal biocatalysts used in energy generation primarily increase the rate of electron transmission due to increased fungal hyphae networking and thereby produce stable electricity which may contribute to external electrochemical operations. Due to this unique property, yeast and other fungi have been preferred over prokaryotes like bacterial cells for electricity generation and wastewater treatment ( Sayed and Abdelkareem, 2018 ). Fungal cells are not only useful in the production of bioelectricity and wastewater treatment processes, but in quality biofuel production as well. Fungal species such as Rhodosporidium toruloides, Yarrowia lipolytica, Cryptococcus sp., Aspergillus sp., Penicillium sp., and Trichoderma reesei play an important role in biofuel production. Biodiesel production using microbial lipids is popularly known as single cell oils (SCOs), that, has attracted the immense attention of researchers. Huang et al. (2009) reported microbial oil production from sulphuric acid-treated rice straw hydrolysate (SARSH) by cultivation of Trichosporon fermentans . Few studies have recorded the potential of white-rot fungus and soft rot fungus in the degradation of lignocellulosic materials ( Anderson and Akin, 2008 ; Sun and Cheng, 2002 ). There are reports on the existence of endophytic microorganisms in some oleaginous plants (including their seeds) that have a capacity for microbial lipid production ( Peng and Chen, 2007 ; Dey et al., 2018 ). Gliocladium roseum , an endophytic fungus, is known for its ability to commercially produce biodiesel (mycodiesel) from a variety of substances. Because of its low carbon content, biodiesel produced by yeast or other fungi is more environment friendly and of high value. Recently, metabolic engineering has strived to increase the synthesis of lipids in fungal cells. In S. cerevisiae strain YPH499, glyceraldehyde-3-phosphate pathway is modified by over-expressing genes namely - glycerol kinase, diacylglycerol acyltransferase and phospholipid diacylglycerol acyltransferase . The alteration of genetic makeup resulted in the accumulation of large number of TAGs in that particular strain ( Yu et al., 2013 ). Besides, metabolic engineering, global transcription machinery engineering (GTME), enzyme engineering and metagenomics may be exploited together to manipulate cells in order to improve the target strain and its performance in energy generation."
} | 2,854 |
32466455 | PMC7361963 | pmc | 4,193 | {
"abstract": "Polyesters based on 2,5-furandicarboxylic acid (FDCA) are a new class of biobased polymers with enormous interest, both from a scientific and industrial perspective. The commercialization of these polymers is imminent as the pressure for a sustainable economy grows, and extensive worldwide research currently takes place on developing cost-competitive, renewable plastics. The most prevalent method for imparting these polymers with new properties is copolymerization, as many studies have been published over the last few years. This present review aims to summarize the trends in the synthesis of FDCA-based copolymers and to investigate the effectiveness of this approach in transforming them to a more versatile class of materials that could potentially be appropriate for a number of high-end and conventional applications.",
"conclusion": "9. Concluding Remarks Sustainability has become an integral part of polymer science and will remain in the forefront of research and development of new biobased plastics. Polymers derived from FDCA are expected to play a leading role in the following years as part of the bioeconomy initiative that is promoted nearly worldwide. To ensure the reduction in the use of fossil-based plastics and the accumulation of their waste, authorities have to support research organizations and industries both financially but also with legislation and by educating the public, while keeping in mind the ultimate goal of stabilizing the atmospheric greenhouse gas levels and putting a halt on global climate change. The dominance of biobased polymers will depend heavily on the advances on lignin valorization and isolation of high-purity monomers that will allow the production of cheap, colorless plastics. After reviewing the available literature, copolymerization is clearly a valuable method for the tuning of the properties of FDCA-based polyesters. They can be comparable or even better than commercial, fossil-based polymers in terms of physicochemical properties. This will allow their use in diverse applications that can extend further from specialty packaging. Simultaneously, copolymerization helps with overcoming some of the problems that are related with FDCA, such as coloration, high cost, and a lack of biodegradation. Some aspects that must not be overlooked are the methods of waste disposal of bioplastics, their effect on microplastic formation in the oceans, and life cycle assessments. Biodegradation studies that simulate the environment only concern soil, but as the severity of the accumulation of polymeric microplastics in water masses is recognized, the degradation of new biobased polymers in aquatic environments will have to be evaluated, too. In this direction, new testing standards need to be implemented. Several research groups from all over the world have published valuable data on a plethora of copolyesters that are synthesized in the typical polyester synthesis infrastructures. Hopefully, this work will help accomplish the dream of a sustainable future that will require a close collaboration of industries, scientists, and governments.",
"introduction": "1. Introduction As the worldwide waste accumulation keeps increasing and the fossil-based energy sources and chemicals are rapidly depleting, governments, industries, and academia have turned their focus on developing methods to optimize the exploitation of natural resources toward a sustainable ‘green’ future. According to the European Commission (EC), bioeconomy is “the production of renewable biological resources and the conversion of these resources and waste streams into value-added products, such as food, feed, bio-based products and bioenergy” [ 1 ]. The EC has set a specific strategy on bioeconomy that includes an increase in the funding for research and innovation and scaling-up of the biobased industrial sector [ 2 ]. This strategy is reflected on the €3.7 billion public–private partnership, the Bio-Based Industries Joint Undertaking, which operates under Horizon 2020 [ 3 ]. Since the 1950s, the use of plastics dominates everyday life, and it is estimated than in 2050, the global cumulative plastic waste generation will exceed 25 billion tons, of which an impressive 12 billion tons will end up either in landfills or in the environment, and only a meager 9 billion tons will be recycled [ 4 ]. The effect of petrochemical-based plastics on the environment is multifaceted; it includes the depletion of petrochemical resources, the increase of the atmospheric CO 2 levels, and the rapid accumulation of waste in both land and oceans. In this light, great efforts have been undertaken to replace conventional plastics with new, sustainable biobased plastics synthesized with monomers derived from biomass. The effective isolation of renewable monomers and the large-scale synthesis of their corresponding plastics is an area where both academia and industry have been focusing on during the last few years. One of the most interesting monomers derived from biomass is 2,5-furan dicarboxylic acid (FDCA), which is an oxidation product of furfural that is included in the top value-added chemicals from biomass list as compiled by the US Department of Energy [ 5 ]. Its importance arises from its chemical structure, as it contains a rigid furan ring and two di-acidic side chains that can easily yield condensation polymers, similarly to terephthalic acid (TPA). Not surprisingly, many companies are therefore either focused on producing or are planning to produce FDCA from biomass in the near future (e.g., Avantium, Novamont, AVA Biochem, Origin Materials, Corbion) or its dimethylester dimethyl furan dicarboxylate (DMFD) (DuPont) [ 6 ]. A plethora of polymers can be synthesized starting from FDCA such as polyesters, polyamides, polyurethanes, and thermosets [ 7 ]. As a result of its similarity with TPA, FDCA-based polyesters are assumed to be the biobased homologues of highly-popular TPA-polyesters such as poly(ethylene terephthalate) (PET), poly(propylene terephthalate) (PPT), and poly(butylene terephthalate) (PBT). Poly(ethylene 2,5-furan dicarboxylate) (PEF) is the most important polyester derived from FDCA due to its similarity with PET, and it is expected to start being commercialized in 2023 [ 8 ] and reach a market value of $129.3 million by 2025 [ 9 ]. In contrast with most Europe-based companies that are planning on commercializing PEF, DuPont has turned its focus on poly(propylene 2,5-furan dicarboxylate) (PPF) [ 10 ], most likely for fiber applications. In addition to their renewable nature, both polyesters have better mechanical, barrier, and thermal properties than their TPA homologues PET and PPF [ 11 ], as reported by a large body of literature [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. The implementation of two big European projects, namely PEFerence (No 744409) funded by Horizon2020 and the COST action FUR4Sustain (CA18220) will boost innovation, aiming to overcome current obstacles and to push toward the commercialization of FDCA-based polyesters. Regardless of their great potential as biobased polymers, FDCA-based polyesters have their limitations. A number of them display slow crystallization rates, a lack of biodegradability, and high rigidity and fragility, which can limit their overall use. Numerous researchers have applied the method of copolymerization to modify the properties of FDCA-based polyesters with a variety of cyclic or aliphatic diols and/or diacids. A summary of the copolyesters reported in the open literature is presented in Table 1 . Nowadays, various copolyesters such as poly(butylene adipate- co -terephthalate) (PBAd- co -PBT), poly(1,4-cyclohexanedimethylene terephthalate- co -isophthalate) (PCHDMT- co -PCHDMI), poly(ethylene- co -1,4-cyclohexanedimethylene terephthalate) (PET- co -PCHDMT), poly(ethylene terephthalate- co -glycolate) (PET- co -PEG), poly(lactide- co -glycolide) (PLA- co -PGA) find applications in specialty packaging, agriculture, and medicine. The comonomers used provide either improved or new properties depending on the final application. For example, adipate units in PBAd- co -PBT copolymers (Ecoflex ® , Origo-Bi) provide them with biodegradability [ 20 ]. Commercial products of copolyesters include Tritan ® , Glass Polymer ® , Eastar ® , Vistel ® (thermoplastic resin), Dynacoll ® S, Petaflex TM , and others. Therefore, it may be of no surprise that copolymerization, as a well-known, widely applied designing method of polymers with tunable properties, is also expected to be applied on FDCA-based polyesters. A small number of patents has already been filed concerning FDCA-based copolyesters with pryomellitic dianhydride, pentaerythritol and their combinations [ 21 ], polyethers [ 22 ], isosorbide [ 23 ] and bis(hydroxymethyl)cyclohexane (cis, trans or both), 2,2-dimethyl-1,3-propanediol (PD), poly(ethylene glycol) (PEG), poly(tetrahydofuran), glycerol, pentaerythritol, lactic acid, 6-hydroxyhexanoic acid [ 24 ]. The literature on polymers with furan rings has been initially reviewed in 2009 [ 25 ], later on with a focus on polyesters in 2013 [ 11 ], in 2016 by our group [ 26 ] and more recently only a brief review on the progress of FDCA-based polyesters was published [ 27 ]. As of 2016, only a few publications on FDCA-based copolyesters were available. Since then, the number of publications and citations grew rapidly, revealing the increased scientific interest on this topic. It is also noteworthy that only a limited number of patents on FDCA-based copolyesters is available up to date [ 21 , 22 , 23 , 24 ]. The aim of this review article is to collect and sum up all the information provided by the published literature on FDCA-based copolyesters, with focus on the tuning of the properties depending on the type of comonomers used, as well as their potential applications."
} | 2,460 |
26583519 | PMC7571853 | pmc | 4,194 | {
"abstract": "Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.",
"conclusion": "Conclusions The advent of omics technologies, coupled to heightened interest in biofuels motivated by the drive towards a sustainable energy future, has driven a rapid increase in our repertoire of lignocellulose-active genes and understanding of natural paradigms. Furthermore, recent discoveries in polysaccharide oxidation [ 11•• ], substrate binding paradigms [ 65• ], enzyme domain architectures [ 8• , 9• ], synergies between enzymatic modes of action [ 66 ] and enzymes for lignin bond cleavage [ 28• ] highlight the fact that many discoveries remain ahead of us. Our understanding of the deconstruction process at molecular and microscopic levels has been enhanced by innovative visualisation of degradation of experimental substrates [ 8• , 67 ]. However, the development of detailed sequence–structure–function relationships for individual enzymes still lags behind, even for enzymes that are considered to be well characterised, such as fungal cellulases [ 3•• ] and hemicellulases [ 68• ], and certainly in more recently discovered oxidative enzymes [ 11•• ] and those involved in lignin degradation [ 15 ]. Beyond understanding single enzymes, the ability to understand how cocktails of enzymes work together synergistically will be undoubtedly crucial to understanding how to harness paradigms observed in Nature and to optimize these to industrial conditions. The ability of organisms and microbial communities to adjust their enzyme cocktails to different substrates almost certainly contains some clues. Tolerance to specific conditions may guide selection of enzymes for biotechnological exploitation [ 59• ]. A more complete understanding and exploitation of the evolutionary inventions offered by the Tree of Life to overcome recalcitrance will ultimately be achieved by combining tools from diverse fields including microbiology, zoology, biochemistry, omics approaches, synthetic biology, advanced imaging and substrate characterisation [ 3•• ].",
"introduction": "Introduction Land plants direct most photosynthetically fixed carbon into lignocellulose, a composite of the polymers cellulose, hemicellulose, pectin and lignin. During the life of the plant, this complex matrix provides structural integrity, and resistance to herbivores and pathogens, so most lignocellulosic biomass is processed by saprophytes and detritivores in detrital food webs. Biomass can be used as a feedstock for biofuel generation, but is recalcitrant to enzymatic processing due to barriers to enzyme access that arise from the paracrystallinity of cellulose, the complexity of the hemicellulose coating of cellulose microfibrils, and the interpenetration and encapsulation of polysaccharide components by lignin. In industrial processes, recalcitrance is overcome by severe chemical and physical pre-treatments, but organisms achieve lignocellulose deconstruction under physiologically tolerable conditions. To assist the prospecting of biodiversity for lignocellulolytic mechanisms with potential for biotechnology applications, a discussion meeting was held in September 2013 at the Linnean Society in London, which reviewed the vast array of mechanisms across the Tree of Life. This article captures and updates the diverse chemical and organismal perspectives brought to the subject by the participants in the meeting."
} | 1,115 |
40301238 | PMC12040808 | pmc | 4,195 | {
"abstract": "To advance the utilization of microbial cell factories in gas fermentation processes, their physiological and biotechnological characteristics must be understood. Here, we report on the construction and operation of a novel device, the Gas and Pressure Controller (GPC), which is specifically designed for the automated control of the headspace gas pressure of closed cultivation bottles and facilitates automated gassing, sparging, monitoring and regulation of the headspace volume operated in closed batch cultivation mode in real time. As proof of concept, the physiological and biotechnological characteristics of four autotrophic, hydrogenotrophic methanogenic archaea were examined to quantify novel physiological limits through the elimination of gas limitation during growth and methane formation. We determined unprecedented high maximum specific methane productivity (qCH 4 ) values for: Methanothermobacter marburgensis of 169.59 ± 12.52 mmol g − 1 h − 1 , Methanotorris igneus of 420.21 ± 89.46 mmol g − 1 h − 1 , Methanocaldococcus jannaschii of 364.52 ± 25.50 mmol g − 1 h − 1 and Methanocaldococcus villosus of 356.38 ± 20.79 mmol g − 1 h − 1 . Obtained qCH 4 of M. marburgensis is more than 10-fold higher compared to conventional closed batch cultivation set-ups and as high as the highest reported qCH 4 value of M. marburgensis from fed-batch gas fermentation in stirred tank bioreactors. Furthermore, the GPC demonstrated reliable functionality with Methanococcus maripaludis , operating safely and autonomous during long term cultivation. This novel device enables optimal headspace pressure control, providing flexibility in application for various gas-fermenting biotechnological processes. It facilitates near optimal cultivation conditions in semi-continuous closed batch cultivation mode, the analysis of limiting factors in high-throughput experimental design and allows for automated biomass production of autotrophic, hydrogenotrophic methanogens. Graphical abstract \n \n Supplementary Information The online version contains supplementary material available at 10.1186/s13568-025-01872-y.",
"introduction": "Introduction In time of limiting resources, microbiology and biotechnology research avenues focus on establishing sustainable production processes. A complete transition towards sustainability may not be solely achieved through one technology but requires a synergy of multiple technologies from different industrial sectors and advances from various scientific fields. Two promising and rapidly emerging biotechnological fields are gas fermentation (Liew et al. 2022 ; Woern and Grossmann 2023 ) and archaea biotechnology (Straub et al. 2018 ; Pfeifer et al. 2021 ). Archaea are a group of prokaryotic microorganisms that exhibit unique biochemical, metabolic and physiological properties that allow application in a wide variety of industrial applications (Pfeifer et al. 2021 ; Carr and Buan 2022 ; Aparici-Carratalá et al. 2023 ). Despite their physiological and biotechnological potential, archaea remain largely underexplored in both scientific research and technological applications. However, many different compounds can be nowadays produced by archaea (Pfeifer et al. 2021 ; Rittmann et al. 2021 ) and major advancement in archaea biotechnology have been recently achieved (Rittmann et al. 2023a , b , c , d ), with a few archaeal production processes currently being scaled-up beyond lab-scale (Pfeifer et al. 2021 ). Significant improvements using archaea have also been made in the context of gas fermentation processes, converting gaseous C1 compounds into gaseous or liquid compounds. In this regard, biohydrogen production through the water gas shift reaction (Kim et al. 2013 , 2017 ; Rittmann et al. 2015b ) as well as the conversion of carbon dioxide (CO 2 ) and molecular hydrogen (H 2 ) into methane (CH 4 ) (Seifert et al. 2014 ; Rittmann et al. 2018 ; Mauerhofer et al. 2021 ) have been thoroughly studied. These bioprocesses may act as carbon utilization avenues, reducing global carbon emissions by converting inorganic carbon into organic material through autotrophic organisms. Methanogenic archaea (methanogens) (Lyu et al. 2018 , 2022 ) are anaerobic organisms and possesses ecological flexibility that allows them to inhabit various anoxic environments including the gastrointestinal tract (Borrel et al. 2023 ) and could possibly thrive in extraterrestrial environments (Taubner et al. 2018 ). They utilize a wide range of different substrate types including methyl compounds, acetate and C1 compounds (Kurth et al. 2020 ). Autotrophic, hydrogenotrophic methanogens are archaeal cell factories for H 2 /CO 2 to CH 4 conversion (Rittmann et al. 2014 , 2018 ; Rittmann 2015 ) and may be employed for proteinogenic amino acid production (Rittmann et al. 2023a , b , c , d ; Taubner et al. 2023 ; Reischl et al. 2025 ). However, a major obstacle that remains is to characterize methanogens in high-throughput screening settings– before bioprocess development shall be initiated. Examining the physiological boundaries of methanogens under comparable and highly reproducible cultivation conditions is therefore essential for identifying and prioritizing strains for their subsequent optimization. Several studies already emphasize the importance of bioprocess factors for regulating the physiological output of methanogens, including gassing rates, pH, stirring rates, temperature, media composition and dilution rates in various cultivation settings (Seifert et al. 2014 ; Rittmann et al. 2015a , 2018 ; Pappenreiter et al. 2019 ; Mauerhofer et al. 2021 ). Anaerobic cultivation of gas-utilizing microorganisms must meet specific requirements, including the prevention of molecular oxygen (O 2 ) exposure, as O 2 is toxic to varying degrees for the strict anaerobic organisms (Mauerhofer et al. 2019 ; Hanišáková et al. 2022 ). The most used cultivation technique in anaerobic microbiology and biotechnology laboratories is closed batch, commonly performed in serum bottles (Taubner and Rittmann 2016 ; Mauerhofer et al. 2019 , 2021 ; Hanišáková et al. 2022 ) and, in the case of autotrophic, hydrogenotrophic methanogens, under slight overpressure conditions (Taubner and Rittmann 2016 ). Later, scale-up of bioprocesses occurs in bioreactors, during which continuous supply of gaseous substrates in fed-batch (Abdel Azim et al. 2017 ) or continuous culture mode (both liquid and gas feed) is employed (Seifert et al. 2014 ). The bottleneck in closed batch, but also in bioreactors, is (usually) not the catalysis rate of the biomass, but the gas transfer from the gas into the liquid phase. It is therefore necessary to optimize the gas transfer rate (GTR) using biochemical bioengineering methods (Rittmann et al. 2015a ; Takors et al. 2018 ). This can be achieved by increasing the partial pressure of the respective gas, which is why gas fermentations are specifically carried out under elevated pressure (Takors et al. 2018 ; Taubner et al. 2018 ; Pappenreiter et al. 2019 ; Mauerhofer et al. 2021 ), or though increasing the gas-liquid mass transfer coefficient (k L a) (Takors et al. 2018 ). Cultivation in closed batch cultivation mode is usually performed before process development is initiated or for obtaining seeding cultures for inoculation (Rittmann et al. 2015a ). This is due to the cost-effectiveness, low expertise requirements of operators and suitability for high-throughput experiments compared to operating bioreactors. Successful outcomes of closed batch cultivations include the assessment of physiological parameters by investigating the methane evolution rates of autotrophic, hydrogenotrophic methanogens (Mauerhofer et al. 2021 ), studies of analysis of growth behavior under elevated heavy metals and volatile fatty acids concentration (Abdel Azim et al. 2018 ) as well as media optimization studies (Abdel Azim et al. 2017 ; Mauerhofer et al. 2021 ). Furthermore, closed batch cultivations are an integral part of biomass production pipelines (Palabikyan et al. 2022 ). However, during closed batch mode, sub-optimal supply of the gaseous substrate prohibits balanced growth shifting the growth kinetics to a disproportionate pattern, labelling it unsuitable for modelling and scale up of biomethanation processes. Therefore, optimizing cultivation conditions in closed batch setups is crucial to be able to draw significant conclusions in screening experiments, even in a small-scale high-throughput design. In this article, we present the functionality and operation of an innovative device, designed for automated gassing, purging, sparging and pressurizing of sealed microbial cultivation bottles independent of the process volume for closed batch cultivations– the Gas and Pressure Controller (GPC). This novel apparatus allows for continuous real-time pressure monitoring throughout the cultivation process. In this proof-of-concept study, convenient quantification of gas conversion and CH 4 production kinetics of five autotrophic, hydrogenotrophic methanogens is demonstrated. The utilization of the GPC facilitates the application of a specific stoichiometry during gas conversion according to the metabolic requirements of the organisms, which leads to a noticeable pressure drop during gas fermentation. The GPC overcomes gas limitations by constant and automated adaptation of the headspace gas pressure, therefore allowing optimal growth in unimpeded conditions. This combination of the advantages of closed batch gas fermentation under gas-unlimited conditions was previously not possible and represents a major leap forward in closed batch cultivation techniques.",
"discussion": "Discussion Set-up constraints of the GPC The quantification of CH 4 production kinetics is based on its partial pressure and can be calculated from the pressure kinetics apparent during methanation (Taubner and Rittmann 2016 ). Ensuring the gas tightness of the system is therefore of utmost importance and regular testing for the closure of the system is highly advised. Inaccuracy may arise through additional pressure loss, facilitated by damage to the rubber stopper. Excessive shaking can subject the needle to extreme stress, potentially causing breakage or wear of the rubber stopper. To mitigate possible risks, only new rubber stoppers were used. Due to the high cultivation temperature and the high re-gassing rate in some experiments, water vapor may form and accumulate within the pipes during the experiment. To prevent any liquids from entering the valves or the manometer, it is crucial to design the length of the tubes in a manner that allows condensation of water vapor to occur within the tubes. An attached sterile filter upstream of the cultivation bottle led to clocking due to vapor formation and deterred gas stream and pressure monitoring. However, sterile filters were installed during cultivation under mesophilic conditions, due to longer tubing and lower temperatures. To further facilitate sterile conditions, the tubing can be removed and autoclaved prior to conducting the experiments. In order to achieve an automated operation that ensures safe release and re-pressurizing of cultivation bottles, unwanted contact between the needle and liquid has to be avoided. This can occur frequently during heavy shaking and significantly increases the risk of culture being drawn into the system, leading to subsequent contamination and potential damage. A significant improvement would be the combination with a non-invasive OD sensor, which would eliminate processing time when measuring biomass concentrations. Data evaluation and uncertainty analysis The application of several data filtering steps was successful in eliminating background data, present during the pressurizing phase at the beginning of each cycle. Due to the dissolution of gases into the liquid phase, a strong aberrant pressure loss occurs at the beginning of the cultivation cycles, which must be mitigated, due to an exaggerated CH 4 production visible as sharp spikes in the MER plots. More rigorous data trimming results in greater data loss and must therefore be adjusted individually based on the organism and the experimental goals. However, preserving the complete cultivation duration is of minor importance when comparing physiological parameters. The methanation kinetics and biomass production kinetics must be evaluated separately, as the actual cultivation time differs from the cycles determined by the algorithm due to data trimming and data processing. Therefore, the CH 4 production kinetics and the timepoint of biomass monitoring do not align accurately. The time intervals for calculating the growth rate were determined manually, based on the assumption that no significant metabolic activity occurs during pause of the GPC and the subsequent shutdown of the shaker. This is evident from the short pressure interval right before shaking and the GPC begins monitoring the headspace pressure. This pressure remains stable when cultivating M. marburgensis , while most cycles of the hyperthermophiles show an increase, presumably because the process temperature has not yet been reached. However, shaking rapidly induces biomethanation and biomass production suggesting that the discussed time interval can be neglected. Comparison of growth and CH 4 production kinetics Four different autotrophic, hydrogenotrophic methanogens were chosen and are among the highest performing CH 4 producers so far identified (Mauerhofer et al. 2021 ). M. marburgensis and M. jannaschii additionally exhibits a broad range of fed-batch or continuous culture cultivations data (Mukhopadhyay et al. 1999 ; Seifert et al. 2014 ) or possess genetic tools (Susanti et al. 2019 ; Fink et al. 2021 ), while biotechnology and physiological studies concerning M. igneus and M. villosus are scarce. Many gas fermentation bioprocesses are inhibited by either liquid or gas limiting conditions, a situation particularly present during closed batch cultivations. To improve GTR into the liquid phase an enlargement of the gas-liquid boundary is required, frequently achieved by rigorous stirring or shaking, or by increasing the driving force through applying barophilic conditions (Seifert et al. 2014 ; Takors et al. 2018 ; Pappenreiter et al. 2019 ; Mauerhofer et al. 2021 ). In order to push the metabolic activity of observed organism to the limits, even in closed batch cultivation mode, the unidirectional shaking was set to maximum while the GPC provides excess of gaseous substrate. During the experiments, no gas limitation was observed, as indicated in the acceleration of the conversion rate until an inflection point, defined as the timepoint where maximum conversion (k min / bar h − 1 ) and MER max occurs, is reached (Pappenreiter et al. 2019 ). It was not possible to measure the k L a using established methods, as the gassing in and gassing out method with O 2 is only applicable in fed-batch fermentation with the appropriate probe, preferable with a short response time. Although probes for measuring H₂, CO₂, or CH₄ are available, they are not commonly used in closed batch systems. The k min determined for all experiments were around two to four times (2.8 to 4.1 bar h − 1 ) higher than the previously inspected values at 10 bar (max 1.52 ± 0.27 bar h − 1 ) and challenging most maximum conversion rates at 50 bars (0.85 ± 0.64 to 5.11 ± 0.55 bar h − 1 ) (Mauerhofer et al. 2021 ). It should be noted that our system allows for GTRs that are comparable to, or higher than those achieved in high-pressure experiments. However, making a precise statement about physiological properties was not possible, as the biomass was omitted during the analysis. The inflection point, signaling the peak in MER, marks the precise moment when a putative liquid limitation occurs and, except for M. igneus , where one inflection point is off by one cycle, remains consistent within the experimental group. M. marburgensis shows optimal performance for about 7.06 h, in contrast to hyperthermophilic methanogens that reach the inflection point earlier, after 3 h for M. igneus , 3.3 h for M. villosus , and 3.7 h for M. jannaschii . Additionally, M. igneus and M. jannaschii show a sudden decrease in MER after reaching liquid limitation. Catalytic inhibition in M. marburgensis arises later, coinciding with a stable qCH 4 up to that point. The hyperthermophilic methanogens decrease in qCH 4 highlight early limitation right from the start of the cultivation and review different degrees of optimization potential of cultivation media and show specific differences between thermophilic and hyperthermophilic organisms. While the media of M. marburgensis was intensely optimized regarding trace elements (Schönheit et al. 1980 ; Abdel Azim et al. 2018 ), the hyperthermophilic 282c media was mostly unchanged or had been substituted with yeast extract or peptone for heterotrophic growth (Topçuoğlu et al. 2019 ), therefore compulsory inhibition of methanogenesis arise early. However, a thorough analysis of the limiting factors requires investigating the specific media requirements and conducting experimental testing of methanation under conditions of limitation or saturation. Previous studies highlight the correlation between trace elements on biomethanation efficiency (Abdel Azim et al. 2017 ; Rittmann et al. 2018 ). A limitation in trace element is therefore probable, deriving from lower absolute amounts and potentially higher demands and specificity, but might only explain the limitation localized by the inflection point. Additionally, the biological availability of chloride versus sulfate-based media needs to be verified. The necessity of Selenium (Se) for growth of methanogens was also reported, due to Se being part of enzymes responsible for energy metabolism (Grahame and Stadtman 1993 ). It can be assumed that selenite concentration is underrepresented, as higher concentrations were shown to be beneficial (Mukhopadhyay et al. 1999 ). The trends of qCH 4 could also be a consequence of carbon regulation. The experiments indicate that during the initial phase of cultivation, the carbon flux shifts toward biomass production and shows a reversed trend after reaching the inflection point (Fig. 3 ). Therefore, the qCH 4 decreases as the carbon flux in the biomass increases. This goes along with the theory that during sufficient nutrient supply, biomass growth is increased while the metabolism is less efficient. Conversely, under substrate limitation, growth decreases, but metabolic activity increases (Molenaar et al. 2009 ). This effect is more pronounced during hyperthermophilic growth, as during cultivation of M. marburgensis , where Y CH4 at the inflection point correlates with qCH 4max , both exhibiting a positive trend that contradicts the proposed carbon flow regulation. Another possible explanation could be the instability of hyperthermophilic growth, where the rapid turnover leads to quick cell division and lysis, resulting in fewer enzymatically active cells overall. Furthermore, the pH was not measured during the experiment, as measuring pH in a closed environment is a highly inversive process. This is a known problem during closed batch gas fermentations and is extra challenging in anaerobic environments. An investigation of pH values under elevated pressures illustrates the decrease in CO 2 solubility under elevated temperatures. It can therefore be assumed that the influence of the pressure as well as the CO 2 solubility and consumption on the pH is limited under thermophilic and hyperthermophilic conditions. The highest observed specific growth rate of M. marburgensis (µ max = 0.55 ± 0.06 h − 1 ) was lower than the previously proposed (Schönheit et al. 1980 ) and confirmed (Abdel Azim et al. 2017 ) optimum of 0.69 h − 1 . However, earlier experiments were carried out under optimized conditions in bioreactors (Abdel Azim et al. 2017 ). The qCH 4max of 169.59 ± 12.52 mmol g − 1 h − 1 is, under consideration of the standard deviation, almost equal to the maximum reported qCH 4 (Abdel Azim et al. 2017 ), which is another indicator that the system during that time is not limited by the gas transfer or by liquid media depletion but by the physiology of the organism itself. Ascertained qCH 4max from M. igneus , M. jannaschii , M. villous were the highest ever recorded of any methanogen only challenged by Peillex et al. (Peillex et al. 1990 ). Here, reported qCH 4 values exceed other published data by several factors, along with significant variations, potentially not reflecting actual physiological limits of M. marburgensis (Rittmann et al. 2018 ). The newly determined specific growth rates of the hyperthermophilic methanogens were, to our research, additionally the highest that had ever been reported (Miller et al. 1988 ; Mukhopadhyay et al. 1999 ; Topçuoğlu et al. 2019 ; Mauerhofer et al. 2021 ). Investigating qCH 4 , MER, µ and Y CH4 shows lack of homogeneity in some cycles. Therefore, the statistical analysis was not conducted with the complete data set. Statistical relevance was found in the first cycle of qCH 4 between M. marburgensis and the hyperthermophiles but not between the hyperthermophilic organisms themselves. This highlights the methanation capacities between organisms of different temperature regimes. The biomethanation capacities of tested hyperthermophiles are probably too closely related to detect meaningful differences. Long term cultivation with M. maripaludis M. maripaludis is one of the best studied autotrophic, hydrogenotrophic methanogens and exhibit an advanced molecular toolkit, including CRISPR-mediated genome editing, that labels the organism as a valuable cell factory well-suited for physiological studies and the production of a wide range of different products (Walters et al. 2011 ; Richards et al. 2016 ; Bao et al. 2022 ; Li et al. 2022 ; Xu et al. 2023 ; Du et al. 2024 ). As a result, increased efforts have been made towards improving biotechnological capability, such as in the production of biomass (Palabikyan et al. 2022 ). M. maripaludis was successfully cultivated over 80 h while constant monitoring and adjusting of the headspace pressure. Figure 4 and Fig. S13 delineate the gas consumption during cultivation with suboptimal culture to headspace volume ratio. While the exponential increase in MER after the lag phase suggests exponential growth behavior, an inflection point is reached soon after, signaling the transfer from gas unlimited to limited conditions. Afterward, a decreasing trend in the MER, starting anew with each gassing cycle, can be observed. It is assumed that during gas limited conditions and biomass saturation the MER matches the GTR. Since the GTR is the product of the k L a and the driving force (Δc) from the gas to the liquid phase, and a stable k L a is assumed during the cultivation process, the MER can be considered a direct function of the pressure. However, the total CH 4 production decreases with each cycle, accompanied by an increase in cultivation time, evident by the reduced slope and the transition to more curved methanation kinetics over time. This hypothesis requires further testing, as cultivation results obtained from M. maripaludis using CO 2 as carbon source reveal liquid limitations as early as the second cycle. This is evidenced by a decline in MER at consistent pressure levels across successive methanation cycles (Fig. S14 ), a trend also apparent in the wavelike pattern of the total amount of produced CH 4 in mol (Fig. S15 ). In conclusion: The GPC facilitates automated gassing of a wide range of different cultivation bottles in closed batch settings and allows for monitoring and regulation of the headspace gas pressure in real time over virtually unlimited duration. Here, the GPC has been specifically used to elucidate physiological and biotechnological characteristics of autotrophic, hydrogenotrophic methanogens. Further testing is needed to adapt the system for other gas conversion processes, such as those that support microbial activity followed by a fixed stoichiometry. First, the GPC allows quantification of physiological limits of autotrophic, hydrogenotrophic methanogens by eliminating gas limitation during growth of organisms and facilitates near optimal cultivation conditions in closed batch setups. As a result, previously unknown and very high qCH 4 values for three hyperthermophilic methanogens have been determined. Second, the device can accurately determine the occurrence of liquid limitations by visualizing the conversion point of metabolic inhibition. This allows for conclusions about limiting components and can be further exploited by high-throughput experiments. Third, the GPC may act as a tool for generating biomass on a large scale and by reaching specific growth rates that would not be possible in established manual closed batch cultivation settings. Finally, the GPC demonstrates application flexibility towards various applications in the fields of gas fermentation with hydrogenotrophic methanogens and archaea biotechnology."
} | 6,333 |
27840600 | null | s2 | 4,196 | {
"abstract": "Water hampers the formation of strong and durable bonds between adhesive polymers and solid surfaces, in turn hindering the development of adhesives for biomedical and marine applications. Inspired by mussel adhesion, a mussel foot protein homologue (mfp3S-pep) is designed, whose primary sequence is designed to mimic the pI, polyampholyte, and hydrophobic characteristics of the native protein. Noticeably, native protein and synthetic peptide exhibit similar abilities to self-coacervate at given pH and ionic strength. 3,4-dihydroxy-l-phenylalanine (Dopa) proves necessary for irreversible peptide adsorption to both TiO"
} | 156 |
23759552 | PMC4002587 | pmc | 4,199 | {
"abstract": "We have recently identified two genes coding for ammonium transporters (AMT) in Sorghum bicolor that were induced in roots colonized by arbuscular mycorrhizal (AM) fungi. To improve our understanding of the dynamics of ammonium transport in this symbiosis, we studied the transfer of soil-ammonium-derived 15 N to S. bicolor plants via the Glomus mosseae fungal mycelium in compartmented microcosms. The 15 NH 4+ -containing hyphal compartment was inaccessible to the roots in the plant compartment. 15 N label concentrations significantly increased in plant roots and leaves already 48 h after exposure of the AM fungus to the 15 NH 4+ substrate, attesting an efficient symbiotic N transfer between the symbiotic partners and further highlighting that AM symbiosis represents an important component of plant nitrogen nutrition."
} | 209 |
33693714 | PMC9113415 | pmc | 4,201 | {
"abstract": "Abstract The performance of the alkaline fungal laccase PIE5 (pH 8.5) in the\ndelignification and detoxification of alkali-pretreated corncob to produce\nbioethanol was evaluated and compared with that of the neutral counterpart\n(rLcc9, 6.5), with the acidic laccase rLacA (4.0) was used as an independent\ncontrol. Treatment with the three laccases facilitated bioethanol production\ncompared with their respective controls. The lignin contents of\nalkali-pretreated corncob reduced from 4.06%, 5.06%, and\n7.80% to 3.44%, 3.95%, and 5.03%, after PIE5, rLcc9,\nand rLacA treatment, respectively. However, the performances of the laccases\nwere in the order rLacA > rLcc9 > PIE5\nin terms of decreasing total phenol concentration (0.18, 0.36, and\n0.67 g/l), boosting ethanol concentration (8.02, 7.51, and\n7.31 g/l), and volumetric ethanol productivity (1.34, 0.94, and\n0.91 g/l hr), and shortening overall fermentation time. Our\nresults would inform future attempts to improve laccases for ethanol production.\nFurthermore, based on our data and the fact that additional procedures, such as\npH adjustment, are needed during neutral/alkaline fungal laccase treatment, we\nsuggest acidic fungal laccases may be a better choice than neutral/alkaline\nfungal laccases in bioethanol production.",
"conclusion": "Conclusions The potentials of the alkaline fungal laccase PIE5 in the delignification and\ndetoxification of alkali-pretreated corncob to produce bioethanol were evaluated and\ncompared with other fungal laccases, including rLcc9 and rLacA. The laccases\ndecreased phenolic compounds and lignin contents in slurries and improved the\nperformance of S. cerevisiae by shortening the adaptation time and\nenhancing the production rate, cell viability, and volumetric ethanol productivity.\nThe comprehensive performances of rLcc9 were better than that of PIE5, with rLacA\nperformed the best among the three laccases. Based on these data, we concluded that\nacidic fungal laccases may be a better choice than neutral/alkaline fungal laccases\nin bioethanol production.",
"introduction": "Introduction Lignocellulosic feedstocks are low-cost and renewable raw materials that are abundant\nand have no competition on food crops (Balan et al., 2013 ). Bioethanol production from lignocellulosic biomass is\nconsidered promising alternatives to mitigate global climate change and reduce\ndependence on petroleum-based fuels (Fillat et al., 2017 ). However, lignocellulosic materials have a complex and\nrecalcitrant structure. Thus, a pretreatment step is needed to depolymerize\ncellulose, hemicellulose, and lignin to make the biomass more accessible to\nhydrolytic enzymes (Binod et al., 2010 ). Various pretreatment methodologies, including alkaline\npretreatment, acid pretreatment, and their combinations (Pedersen et al.,\n 2010 ), have been developed and applied\nto pretreat a wide range of lignocellulose feedstocks. Compared with acid or\noxidative reagents, the alkaline treatment appears to be the most effective method\nin breaking the ester bonds between lignin, hemicellulose, and cellulose\n(Gáspár et al., 2007 ). Unfortunately, the toxic monomeric lignin compounds released during the lignin\ndegradation process, which comprise phenolic compounds, such as aromatic acids,\ncatechol, 4-hydroxybenzaldehyde, and vanillin (Pedersen & Meyer, 2010 ), as well as furan derivatives and weak\nacids from pentose and hexose sugars degradation, limit the subsequent\nsaccharification and fermentation processes. Different remediation treatments for\ndetoxification, including physical, chemical, and biological treatments, have been\nemployed to reduce the effects of inhibitory compounds (Rodrigues et al.,\n 2001 ; Yang & Wyman, 2008 ; Ranjan et al., 2009 ). However, most chemical and mechanical\nmethods are costly, make the biomass-to-ethanol process more complicated, and\nproduce additional waste products (Liu et al., 2005 ). As an alternative to physico/chemical methods, the\nbiological technology of employing fungal laccase has gained considerable attention\nin the last several years (De La Torre et al., 2017 ; Fang et al., 2015 ; Moreno et al., 2012 , 2015 ; Moreno\net al., 2016 ; Moreno et al.,\n 2013 ; Suman et al., 2018 ). Fungal laccases (benzenediol–oxygen oxidoreductases, EC1.10.3.2) are a family\nof blue multicopper oxidases that can oxidize a wide range of phenolic and aromatic\ncompounds and concomitantly reduce molecular oxygen to water as the only end-product\n(Hoegger et al., 2006 ). Its\ndistinguished oxidative capacity makes fungal laccase a potential tool for modifying\nor partial removal of lignin monomer and its derivatives from the pretreated biomass\nto improve saccharification yields (Fillat et al., 2017 ). Several laccases of fungal origin have been evaluated\non their abilities to delignify and detoxify differentially pretreated materials\nduring the bioethanol production process. Overall, these laccases improved the\nperformances of cellulases and yeast to different extents during saccharification\nand fermentation processes. Fungal laccases have pH optimums at pH 3–5.5 and\nbecome essentially inactive as the pH approaches to neutral and alkaline (Petr,\n 2006 ). Thus, the application potential\nof fungal laccases on bioethanol production was evaluated only at acidic conditions\n(De La Torre et al., 2017 ; Fang\net al., 2015 ; Moreno et al.,\n 2012 , 2015 ; Moreno et al., 2016 ; Suman et al., 2018 ). Due to the fact that very few fungal laccases with optimum pH at\nneutral/alkaline conditions have been reported, the performances of neutral/alkaline\nfungal laccases on the delignification and detoxification of different pretreated\nmaterials during bioethanol production have not been evaluated. By comparison, the\nperformances of several alkaline bacterial laccases, such as that from\n Klebsiella pneumoniae (Gaur et al., 2018 ), Amycolatopsis sp.\n75iv3 (Singh et al., 2017 ), and\n Streptomyces ipomoea (De La Torre et al., 2017 ), have been already evaluated on\ndelignification and detoxification of pretreated materials. rLcc9 is a fungal laccase from Coprinopsis cinerea but expressed in\n Pichia pastoris . It has a pH optimum of 6.5 toward guaiacol (Xu\net al., 2019 ). PIE5 is the first\nfungal laccase with an alkaline pH optimum of 8.5 obtained by the directed evolution\nof Lcc9 at sites E116K, N229D, and I393T (Yin et al., 2019 ). The objective of this study was to assess the\napplication potential of the alkaline fungal laccase PIE5 (pH opt. 8.5) on\nbioethanol production and compare its performance with that of the neutral fungal\nlaccase rLcc9 (pH opt. 6.5) (Xu et al., 2019 ). Simultaneously, a classical acidic fungal laccase rLacA (pH opt.\n4.0) from Trametes hirsuta AH28-2 but expressed in P.\npastoris was used as a separate control (Hong et al., 2006 ). The laccases were employed for the\ndelignification and detoxification of alkali-pretreated corncob. In addition, their\neffects on enzymatic hydrolysis and fermentation were also evaluated.",
"discussion": "Results and Discussion Biochemical Properties of Laccases Three laccases, namely, rLacA, rLcc9, and PIE5, were successfully expressed in\n P. pastoris upon induction with methanol. The proteins were\npurified based on ion-exchange chromatography, as shown by SDS–PAGE and\nnative-PAGE (Fig. 1 ). The purified\nrLacA had optimum pH and temperature of 4.0 and 50°C toward guaiacol,\nrespectively (Hong et al., 2006 ). In addition, rLacA laccase showed a redox potential of\n680 mV. This laccase represents classic fungal laccase, which has optimum\nacidic pH and middle/high-level redox potential. By comparison, the optimum pH\nand temperature of rLcc9 toward guaiacol were 6.5 and 60°C, respectively,\nand those of PIE5 were 8.5 and 60°C, respectively. The redox potentials\nof rLcc9 and PIE5 were 506 and 599 mV, respectively (Yin et al.,\n 2019 ). Compared with classic fungal\nlaccases, these two laccases possess neutral and alkaline pH activities and\nstabilities (Petr, 2006 ). rLcc9 and\nPIE5 showed higher redox potentials (506 and 599 mV) and specific\nactivities (310 and 320 U/mg) compared with their bacterial counterparts (De La\nTorre et al., 2017 ; Gaur\net al., 2018 ; Moreno\net al., 2016 ; Singh\net al., 2017 ), because fungal\nlaccases usually have higher redox potential (470–790 mV vs.\n340–490 mV) and specific activities (>200 U/mg protein\ntoward ABTS vs. <100 U/mg protein) ( http://www.brenda-enzymes.org/ ) (Table 1 ), which ensure the wider substrate\nranges and higher activities of fungal laccases (Pezzella et al., 2015 ; Rodgers et al., 2010 ). Fig. 1. Eelectrophoresis of three purified fungal laccases. (a) SDS–PAGE\nof purified laccases. Lanes: M: protein marker; 1: PIE5; 2: rLcc9; 3:\nrLacA. (b) Native-PAGE of purified laccases. Lanes: 1: PIE5; 2: rLcc9;\n3: rLacA. Staining of laccase activity was performed using 5 mM\nguaiacol as the substrate. Table 1. Summary of Reported Laccases Studied for Detoxification and\nDelignification in Lignocellulosic Ethanol Production Laccase origin Feedstocks Pretreat strategy pH Activities Redox potential Decrease in phenol content Reference Bacterial laccases \n Klebsiella pneumoniae \n Wheat and rice bran Acid 5.0 123 U/mg – – Gaur et al. ( 2018 ) Amycolatopsis sp. 75iv3 Poplar Steam 8.0 – – 15% Singh et al. ( 2017 ) MetZyme Wheat straw Steam 5.5 284 IU/g +450 mV 21% Moreno et al. ( 2016 ) \n Streptomyces ipomoeae \n Wheat straw Steam 8.0 8 IU/ml +450 mV 35% De La Torre et al. ( 2017 ) Fungal laccases Trametes hirsuta AH28-2 Corncob Alkali 4.0 1085 U/mg +680 mV 82% This study \n Coprinopsis cinerea \n Corncob Alkali 6.5 310 U/mg +506 mV 63% This study PIE5 Corncob Alkali 8.5 320 U/mg +599 mV 28% This study \n Trametes villosa \n Wheat straw Steam 4.0 370 IU/ml >730 mV 71% De La Torre et al. ( 2017 ) \n Coriolopsis rigida \n Wheat straw Steam 5.0 49 U/mg – 70–75% Jurado et al. ( 2009 ) \n Ganoderma lucidum 77002 \n Corn stover Steam 5.0 186 U/mg – 84% Fang et al. ( 2015 ) \n Pycnoporus cinnabarinus \n Wheat straw Steam 5.0 60 IU/ml – 67–80% Oliva-Taravilla et al. ( 2015 ) Trametes maxima IIPLC-32 Sugarcane bagasse Acid 5.5 1610 IU/mg – 79.28% Suman et al. ( 2018 ) Chemical Composition of Solid Fractions After Laccase Treatment Alkaline extraction was employed to pretreat the corncob substrate because of its\nhigher efficiency in delignification than other pretreatment strategies\n(Gáspár et al., 2007 ). After alkaline pretreatment, the cellulose proportion of the\nsolid fraction increased (>52.74%) compared with that of the\nuntreated substrate (44.16%) (Table 2 ). This increase was attributed to the extensive\nsolubilization and degradation of lignin and hemicellulose as pointed out by the\nlower proportions of remaining lignin (<7.80% vs. 15.01%)\nand hemicellulose (<28.16 vs. 37.56%, Table 2 ). In accordance with this fact, the\nconcentration of total phenols in the supernatant was 0.98 g/l\n(Table 3 ). These compounds\nare derivatives of lignin (Pedersen & Meyer, 2010 ). Table 2. Composition of Alkaline Pretreatment Corncob Treated With Different\nStrategies Substrates Cellulose (%) Hemicellulose (%) Lignin (%) Ash (%) Other components (%) Crude corncob 44.16 ± 4.12 37.56 ± 0.13 15.01 ± 0.36 1.70 ± 0.33 1.58 ± 0.19 pH 4.0 + C 52.74 ± 0.54 28.16 ± 0.15 7.80 ± 0.26 7.53 ± 0.26 3.77 ± 0.18 pH 4.0 + rLacA 58.65 ± 0.49** 31.10 ± 0.32** 5.03 ± 0.01 5.14 ± 0.19** 0.07 ± 0.00* pH 6.5 + C 53.26 ± 1.07 27.35 ± 0.49 5.06 ± 0.27 6.43 ± 0.14 7.90 ± 0.15 pH 6.5 + rLcc9 59.09 ± 0.16* 30.31 ± 0.22* 3.95 ± 0.04* 5.13 ± 0.28* 1.51 ± 0.06* pH 8.5 + C 55.04 ± 0.90 27.89 ± 0.45 4.06 ± 0.08 5.63 ± 0.14 7.37 ± 0.13 pH 8.5 + PIE5 59.02 ± 1.54 29.59 ± 0.71 3.44 ± 0.02** 4.93 ± 0.09* 3.02 ± 0.14 pH 4.0 + C, NaOH-pretreated corncob with pH\nadjusted to 4.0; pH 4.0 + rLacA, rLacA laccase\ntreatment of (pH 4.0 + C) sample; pH\n6.5 + C, NaOH-pretreated corncob with pH\nadjusted to 6.5; pH 6.5 + rLcc9, rLcc9 laccase\ntreatment of (pH 6.5 + C) sample; pH\n8.5 + C, NaOH-pretreated corncob with pH\nadjusted to 8.5; pH 8.5 + PIE5, PIE5 laccase\ntreatment of (pH 8.5 + C) sample. Results were\ncalculated from two times of independent experiments performed in\ntriplicate ( n = 6). Difference\nin means is significant at the\n* p < .05 or\n** p < .01\nlevel. Table 3. Content of Total Phenols With Different Assay Procedures Substrates After laccase treatment (g/l) Adjusted to pH 4.8 (g/l) After 72 hr saccharification (g/l) Fermentation (2 hr) (g/l) pH 4.0 + C 0.98 ± 0.04 1.09 ± 0.07 1.04 ± 0.07 0.98 ± 0.01 pH 4.0 + rLacA 0.18 ± 0.01*** 0.15 ± 0.00** 0.16 ± 0.02** 0.16 ± 0.02*** pH 6.5 + C 0.97 ± 0.02 0.86 ± 0.00 0.78 ± 0.00 0.77 ± 0.01 pH 6.5 + rLcc9 0.36 ± 0.01*** 0.34 ± 0.00*** 0.29 ± 0.00*** 0.29 ± 0.00*** pH 8.5 + C 0.93 ± 0.05 0.83 ± 0.04 0.74 ± 0.04 0.74 ± 0.05 pH 8.5 + PIE5 0.67 ± 0.05* 0.62 ± 0.11 0.49 ± 0.04* 0.51 ± 0.07 pH 4.0 + C, NaOH-pretreated corncob with pH\nadjusted to 4.0; pH 4.0 + rLacA, rLacA laccase\ntreatment of (pH 4.0 + C) sample; pH\n6.5 + C, NaOH-pretreated corncob with pH\nadjusted to 6.5; pH 6.5 + rLcc9, rLcc9 laccase\ntreatment of (pH 6.5 + C) sample; pH\n8.5 + C, NaOH-pretreated corncob with pH\nadjusted to 8.5; pH 8.5 + PIE5, PIE5 laccase\ntreatment of (pH 8.5 + C) sample. Results were\ncalculated from two times of independent experiments performed in\ntriplicate ( n = 6). Difference\nin means is significant at the\n* p < .05 or\n** p < .01 or\n*** p < .001\nlevel. The water-insoluble fraction of pretreated material is usually separated by\nfiltration, washed with water, and resuspended in a suitable buffer to meet the\npH demand of acidic fungal laccases after alkali pretreatment (De La Torre\net al., 2017 ). In this study, pH\nwas adjusted directly using the mixture as the substrate to avoid the filtration\nand washing steps and decrease operational costs and wastewater. Results from\nthree control groups suggested that the pH adjustment affected the proportion of\ncellulose but not hemicellulose, in which the former decreased (55.04%,\n53.26%, and 52.74%, respectively,\n p < .05) and the latter kept unchanged\n(27.89%, 27.35%, and 28.16%, respectively,\n p > .05) with decreasing pH (8.5, 6.5,\nand 4.0). Along with adjusting pH from extremely alkaline condition to acidic\ncondition by using H 2 SO 4 , the phenolic units of lignin\ncould be polymerized to yield oligomers or undergo grafting reactions onto\npretreated solid fraction, because the H 2 SO 4 used as a\ncatalyst would reduce phenol solubility at harsh conditions (Jurado\net al., 2009 ). As a support to\nthis hypothesis, the lignin content increased remarkably from 4.06% to\n7.8% as the pH decreased from 8.5 to 4.0 (Table 2 ). The changes in concentrations of other toxic compounds, including furan\nderivatives and weak acids, were determined and compared among three control\nsamples. The concentration of formic acid kept unchanged\n(246.2 ± 9.7 mg/l) in the three samples. By\ncomparison, acetic acid concentrations were 114.1 ± 4.6,\n346.8 ± 13.3, and\n413.7 ± 15.1 mg/l in pH 4.0, 6.5, and 8.5 samples.\nSlight decrement for furan derivatives, of which the concentrations were\n127.4 ± 4.9 mg/l (pH 8.5),\n121.8 ± 3.6 mg/l (6.5), and\n120.2 ± 3.1 mg/l (4.0), respectively, was detected\nalong with the pH increment. It was reported that alkaline pH could affect furan\nderivatives’ concentration. For example, in an investigation of\ndetoxification of steam-pretreated wheat straw, the content of 5-HMF and\nfurfural was slightly higher in samples at pH 4 than that at pH 8 (De La Torre\net al., 2017 ). The laccase action on alkali-pretreated corncob results in polymeric lignin\noxidation and/or soluble phenolic compounds oxidation (Kudanga & Le\nRoes-Hill, 2014 ). Furthermore, laccase\ncatalyzed oxidation gives rise to radical species that can evolve degradation\nprocesses, of which oxidative coupling is the primary mechanism resulting in\nhomo- and/or cross-coupling of molecules. Radical species can also act as\nmediators for further oxidation of other compounds, causing bond cleavage, or\nundergo rearrangement per se to result in dead-end products (Pezzella\net al., 2015 ). As a result,\ntreating the pretreated corncob with laccase may cause the reduction of lignin\ncontent. In fact, the lignin content of the pretreated solid fraction decreased\nabout 21.9% and 15.3% after rLcc9 and PIE5 pretreatment\n(Table 2 ). Thus, it was\nreasonable that the cellulose and hemicellulose proportions in\nalkaline-pretreated solid fraction increased by 10.9% and 7.2%,\nand 10.8% and 6.1%, respectively, when compared with their\nrespective controls after the addition of rLcc9 and PIE5. Similar results were\nobtained when alkali-pretreated Brassica campestris straw was\ntreated with laccase from Ganoderma lucidum Tr16 (Yang\net al., 2011 ). By contrast, the\nlignin content of steam-pretreated spruce increased slightly after laccase\ntreatment (Moilanen et al., 2011 ). Redox potential determines the ability and the substrate range of laccase to\noxidize lignin compounds (De La Torre et al., 2017 ). It was reported that the lignin content of unwashed\nsteam-exploded wheat straw had a slight increment after treatment with a fungal\nlaccase from Trametes villosa with high redox potential\n(730 mV) (De La Torre et al., 2017 ). By comparison, lignin content was unchanged when laccases\nwith lower redox potentials (around 450 mV) were employed to treat the\nsamples (De La Torre et al., 2017 ; Moilanen et al., 2011 ; Yang et al., 2011 ). As an independent control in this work, after adding the\nlaccase rLacA, which showed a moderate redox potential of 680 mV, into\nthe alkaline-pretreated corncob, the lignin content decreased 35.5% when\ncompared with its control (Table 2 ). Thus, we suggest that the redox potential of a laccase\ndetermines the change in lignin content after laccase treatment, that is,\nlaccases with high redox potential (e.g., >730 mV) increase lignin\ncontent by catalyzing the grafting and polymerization of most soluble phenolic\ncompounds in samples. By comparison, laccases with moderate and low redox\npotentials (e.g., <710 mV) decrease or do not affect lignin\ncontent because of their limited abilities to oxidize lignin compounds. However,\nmore evidence is needed to prove this hypothesis. The treatment of the alkaline-pretreated solid fraction with rLacA resulted in\nthe highest delignification efficiency (35.5%) among the three fungal\nlaccases when compared to their respective controls. The higher redox potential\nof rLacA (680 mV) compared to rLcc9 (506 mV) and PIE5\n(599 mV) may facilitate the action of laccase toward phenolic units of\nlignin and result in a variety of reactions such as ether and C–C bonds\ncleavage in polymeric lignin ( Moreno\net al., 2016 ). However, after the addition of laccases, rLcc9\nremoved more lignin from the pretreated solid fraction than the PIE5\n(21.9% compared to 15.3%), although the former possessed lower\nredox potential than the later. The difference found in the delignification\nbetween the two laccases could be explained by the sophisticated physicochemical\ncharacteristics of these laccases. For example, PIE5 showed\n K m values of\n3.3 × 10 −4 and\n5.7 × 10 −3 M on guaiacol and\n2,6-DMP, respectively, compared to\n0.9 × 10 −4 and\n2.3 × 10 −3 M of rLcc9 (Yin\net al., 2019 ). Effect of Laccase Treatment on Phenols in Pretreated Solid Fractions Phenol concentrations in pretreated solid fractions with and without laccase\ntreatment were determined and compared with their respective controls\n(Table 3 ). Although the\ntotal phenol concentrations in the rLcc9 and PIE5 control samples were similar,\nit should be noted that the compositions and concentrations of phenols in\nsamples with different pH values were different because alkaline pH can affect\nthe concentrations of furan derivatives and phenols and the solubilization of\nphenolic compounds (De La Torre et al., 2017 ; Jurado et al., 2009 ). As a result, the total phenols decreased by 62.89% (pH\n6.5) and 27.96% (8.5), respectively, after rLcc9 and PIE5 treatment\nbecause of the different affinities of laccases toward different phenolic\ncompounds. As an independent experiment, the total phenols decreased by\n81.63% (pH 4.0) after rLacA treatment (Table 3 ). Several acidic laccases (mainly\nfungal origin) and alkali laccases (mainly bacterial origin) have been evaluated\non their abilities to decrease phenol content. Similar to our results, acidic\nlaccases removed more phenols than alkali laccases regardless of the\nlignocellulose feedstock and pretreatment strategies used (Table 1 ). As shown in these cases, the pH of\nthe slurry and the characteristics of the laccase used can affect the phenol\nremoval from the pretreated lignocellulose. In addition, the redox potential of\nlaccases play a key role in the range of phenol removal (De La Torre\net al., 2017 ). The concentrations\nof total phenols in all the samples did not change too much after adjusting the\npH to 4.8 in this study (Table 3 ). Effect of Laccase Treatment on Enzymatic Hydrolysis of Pretreated\nCorncob Laccase treatment affects the saccharification process. The relative glucose\n(RGR) and xylose concentration recoveries (RXR) in the rLcc9 and PIE5 control\nsamples decreased gradually after 72 hr of saccharification\n(Fig. 2 ). The phenomenon\noccurred probably because of the different degrees of erosion and exposure of\npretreated materials by the harsh conditions when the pH was adjusted to 6.5 and\n8.5 using H 2 SO 4 (Jurado et al., 2009 ; Saha et al., 2005 ). As a support to this conclusion,\nthe surface morphology of the differentially treated corncob was quite different\n(Fig. 3 ). After alkaline\npretreatment, the external surface of the corncob became plicated and slightly\ndehiscent (Fig. 3 ), which resulted\nfrom the removal of lignin and the breakage of the lignocellulosic complex\nconstruction during the pretreatment. Moreover, the toxicity of different\nphenols to cellulosic enzyme activity cannot be ignored (Table 3 ). The saccharification of the rLcc9-\nand PIE5-treated samples showed a substantial increase in RGR and RXR compared\nwith the corresponding controls. The RGR and RXR in rLcc9-treated samples were\nenhanced by 8.81% and 7.00%, respectively, whereas those in\nPIE5-treated samples increased by 5.99% and 5.81%, respectively\n(Fig. 2 ). Saccharification\nefficiency can be affected by many factors, including the phenolic compounds\ncontent, the lignin content, and the available surface area of substrate\n(Fig. 3 ). The increased\nsaccharide recovery after the rLcc9 and PIE5 treatments could be attributed to\nlower lignin content as well as an increase in the porosity and the available\nsurface area. Moreover, according to Palonen and Viikari, laccase treatment\nincreased carboxyl groups of lignin, reducing the hydrophobicity, and increasing\nsurface charge, which led to the decrease of the nonspecific adsorption of\ncellulases to lignin and the improvement of saccharification yields (Palonen\n& Viikari, 2004 ). Fig. 2. RGR(a) and RXR (b) of corncob residues at 72 hr of enzymatic\nhydrolysis after alkaline pretreatment and different laccase treatments\n(white, different laccase treatments; gray, control sample with\ncorresponding pH). rLacA was set as a separate control experiment. Fig. 3. Scanning electron microscopy photomicrograph of the surface of corncob\nsamples. pH 4.0 + C, NaOH-pretreated corncob with\npH adjusted to 4.0; pH 4.0 + rLacA, rLacA laccase\ntreatment of (pH 4.0 + C) sample; pH\n6.5 + C, NaOH-pretreated corncob with pH adjusted\nto 6.5; pH 6.5 + rLcc9, rLcc9 laccase treatment of\n(pH 6.5 + C) sample; pH\n8.5 + C, NaOH-pretreated corncob with pH adjusted\nto 8.5; pH 8.5 + PIE5, PIE5 laccase treatment of\n(pH 8.5 + C) sample. rLacA was set as a separate\ncontrol experiment. Compared with the samples with pH adjusted to 6.5 and 8.5, the surface morphology\nof the sample with pH adjusted to 4.0 seemed to be more plicated and exposed,\nwhich triggered a further increase in the surface area and accessibility of\ncellulose to enzymatic hydrolysis (Fig. 3 ). However, the saccharification of the rLacA-treated sample showed\n3.98% and 2.87% decrements in RGR and RXR, respectively, compared\nwith its corresponding control despite the significant phenolic content\nreduction observed. As ligninolytic enzymes, in addition to soluble phenol\nremoval, laccases have the ability to interact with phenolic units present in\nlignin polymer. Laccase treatment could increase the nonspecific adsorption of\ncellulases to lignin and decrease glucose and xylose yields. In addition,\nTejirian et al. observed that oligomeric phenols formed after the laccase\ntreatment of lignocellulosic materials could inhibit, to a greater extent, the\nenzymatic hydrolysis than single soluble phenols (Tejirian & Xu, 2011 ). Furthermore, a grafting effect may\nexplain the lower saccharification yields of rLacA because it could covalently\ncouple some phenols, such as p -coumaric acid or ferulic acid,\nto the lignin component of the fibers, limiting the accessibility of cellulose\n(Oliva-Taravilla et al., 2015 ).\nMoreover, the formation of phenol-derived compounds by laccase could inhibit\nhydrolytic activities, especially β-glucosidase activity (Ximenes\net al., 2010 ; Ximenes\net al., 2011 ). The enhancement\nof the nonproductive binding of cellulolytic enzymes onto the lignocellulosic\nfibers and a major strengthening of lignin-carbohydrate complexes might be\ninvolved in this reduction. Effect of Laccase Treatment on Cell Viability and Ethanol\nFermentation The viable cells, glucose consumption, and ethanol production of S.\ncerevisiae were improved by PIE5 and rLcc9 treatment compared with\nthe corresponding control sample because of the removal of phenols from the\npretreated substrate (Fig. 4 and\nTable 4 ). However, the level\nof improvement was quite different between samples. During the fermentation\nprocess, the yeast in samples treated with PIE5 or rLcc9 showed a remarkable\nincrease in cell viability, reaching the value of\n0.74 × 10 8 CFU/ml and\n1.33 × 10 8 CFU/ml (Fig. 4 and Table 4 ). Yeast growth increased slower in PIE5-treated samples\ncompared with that in rLcc9-treated samples. The ethanol production rates\nincreased from 0.73 and 0.72 g/l hr in control samples to 0.94 and\n0.91 g/l hr in rLcc9- and PIE5-treated samples, respectively\n(Table 4 ). These increases\nare probably due to the lower phenolic content of PIE5- or rLcc9-treated samples\ncompared with their respective controls. Moreover, the final ethanol\nconcentrations of rLcc9 and PIE5 laccases were 7.51 ± 0.66\nand 7.31 ± 0.54 g/l compared with their respective\ncontrols (7.26 ± 0.23 and\n7.18 ± 0.15 g/l). Several studies have also reported\nan enhancement of the S. cerevisiae performance after\ndetoxification treatments with different laccases. Moreno et al. reported\nhigher glucose consumption rates, ethanol volumetric productivities, and ethanol\nyields when the whole slurry from steam-exploded wheat straw was submitted to\nlaccase treatments and fermented with S. cerevisiae (Moreno\net al., 2012 ; Moreno\net al., 2016 ). Fang\net al. reported that the addition of the G. lucidum \nlaccase Glac15 before cellulase hydrolysis increased ethanol yield by 10%\n(Fang et al., 2015 ). All these\nchanges may be explained by the adaptation of yeast to fermentation conditions,\nwhich depends on different factors, such as the type and concentration of\ninhibitory compounds and their synergistic effects (Klinke et al., 2004 ; Moreno et al., 2013 ; Palmqvist &\nHahn-Hägerdal, 2000 ). Fig. 4. Time course for viable cells (squares), glucose consumption (circles),\nand ethanol production (triangles) during the fermentation of\nhydrolysates from alkali-pretreated corncob residues. (a) PIE5\ntreatment, (b) rLcc9 treatment, (c) rLacA treatment. rLacA was set as a\nseparate control experiment. Table 4. Summary of Saccharification and Fermentation Parameters from Samples With\nDifferent Laccase Treatments Sample EtOH M (g/l) Glu (g/l) Y E/G (g/g) Q G (g/l hr) Q E (g/l hr) Cell viability M \n(CFU × 10 8 per ml) pH 4.0 + C 8.04 ± 0.23 16.90 ± 0.94 0.48 ± 0.02 2.10 † ± 0.12 1.01 † ± 0.09 0.69 ± 0.13 pH 4.0 + rLacA 8.02 ± 0.19 16.23 ± 1.14 0.49 ± 0.01 2.69 § ± 0.19 1.34 § ± 0.06 1.44 ± 0.15** pH 6.5 + C 7.26 ± 0.23 15.88 ± 0.51 0.46 ± 0.03 1.58 ‡ ± 0.05 0.73 ‡ ± 0.06 0.66 ± 0.15 pH 6.5 + rLcc9 7.51 ± 0.16 17.28 ± 0.64 0.43 ± 0.02 2.15 † ± 0.08* 0.94 † ± 0.08 1.33 ± 0.15* pH 8.5 + C 7.18 ± 0.15 14.46 ± 0.21 0.49 ± 0.00 1.44 ‡ ± 0.02 0.72 ‡ ± 0.01 0.49 ± 0.06 pH 8.5 + PIE5 7.31 ± 0.15 15.34 ± 0.63 0.48 ± 0.01 1.89 † ± 0.06* 0.91 † ± 0.06** 0.74 ± 0.08* EtOH M , maximum ethanol concentration; Glu, glucose\nproduced after 72 hr of enzymatic hydrolysis;\n Y E/G , ethanol yield based on total\nglucose content present in the hydrolysate of pretreated corncob;\n Q G , volumetric glucose consumption\nrate based on time when maximum ethanol concentration is achieved:\n6 hr (§), 8 hr (†), and 10 hr\n(‡); Q E , volumetric ethanol\nproductivity based on time when maximum ethanol concentration is\nachieved: 6 hr (§), 8 hr (†), and\n10 hr (‡). Cell viabilityM, maximum viable cells\nduring fermentation. Results were calculated from two times of\nindependent experiments performed in triplicate\n( n = 6). Difference in means\nis significant at the\n* p < .05 or\n** p < .01\nlevel. Yeast cell viability was improved remarkably after treatment with rLacA and\nreached the highest number of 1.43 × 10 8 CFU/ml\nafter 8 hr of fermentation compared with the\n0.69 × 10 8 CFU/ml obtained after\n12 hr of fermentation in control samples. Thus, faster glucose\nconsumption and ethanol production rate were observed. Ethanol productivity\nincreased from 1.01 g/l h at 8 hr in the control samples to\n1.34 g/l h at 6 hr in rLacA-treated samples. These\nincreases are probably due to the lower phenolic content of rLacA-treated\nsamples. Nevertheless, maximum ethanol concentrations\n(8.02–8.04 g/l) and ethanol yields (0.48–0.49 g/g)\nobtained were similar for both control- and rLacA-treated samples\n(Table 4 ) .rLacA laccase\ntreatment did not improve the final ethanol concentration and ethanol yield\n(0.49 g/g, Table 4 ). When\ntreated the steam-exploded whole slurry with T. villosa \nlaccase, faster glucose consumption and ethanol production rates were observed.\nHowever, the ethanol production yield kept unchanged when compared to the\ncontrol (De La Torre et al., 2017 )."
} | 7,588 |
39285903 | PMC11402777 | pmc | 4,202 | {
"abstract": "Summary Energy crops play a vital role in meeting future energy and chemical demands while addressing climate change. However, the idealization of low-carbon workflows and careful consideration of cost-benefit equations are crucial for their more sustainable implementation. Here, we propose tobacco as a promising energy crop because of its exceptional water solubility, mainly attributed to a high proportion of water-soluble carbohydrates and nitrogen, less lignocellulose, and the presence of acids. We then designed a strategy that maximizes biomass conversion into bio-based products while minimizing energy and material inputs. By autoclaving tobacco leaves in water, we obtained a nutrient-rich medium capable of supporting the growth of microorganisms and the production of bioproducts without the need for extensive pretreatment, hydrolysis, or additional supplements. Additionally, cultivating tobacco on barren lands can generate sufficient biomass to produce approximately 573 billion gallons of ethanol per year. This approach also leads to a reduction of greenhouse gas emissions by approximately 76% compared to traditional corn stover during biorefinery processes. Therefore, our study presents a novel and direct strategy that could significantly contribute to the goal of reducing carbon emissions and global sustainable development compared to traditional methods.",
"conclusion": "Conclusion In summary, we identified a simple and viable technology of tobacco utilization to increase the effect of biofuels and bioenergy. By simply autoclaving tobacco leaves in water, a nutrient medium was obtained that readily supports microorganism’s growth and biofuel and biochemical production without additional medium supplements. In addition, our work proposes using global barren/very sparsely vegetated land for growing tobacco to provide a holistic solution for a sustainable global tobacco cultivating expand and provides a reference for policies that address the best use of it for bioenergy by employing LCA to evaluate the environmental impact of tobacco cultivation and industrial utilization.",
"introduction": "Introduction High global demand for fuels and chemicals, coupled with an unstable and uncertain petroleum supply and concerns regarding global climate change, has sparked a renewed interest in renewable alternatives. 1 Energy crops have emerged as sustainable substitutes for petroleum, capable of being converted into various chemicals and fuels that are compatible with existing infrastructure and do not compete with food production. 2 However, the large-scale production of chemicals and fuels from energy crop biomass, rich in cellulose and hemicellulose, necessitates costly processes to break down the complex polymers into their constituent sugars. 3 These processes often involve high temperatures, high pressures, massive chemicals, and expensive enzymes. Harsh processing conditions frequently result in the production of toxic byproducts and the loss of a significant portion of biomass. 4 Furthermore, the total protein content, which accounts for 10%–15% of energy crop biomass, is typically considered waste or requires additional processing for recovery and separate utilization. 5 Additionally, since the hydrolysis products of biomass are rich in sugars but lack other essential elements, additional nitrogen and phosphorus are necessary to support fermentation. 6 Consequently, the widespread use of energy crops as fermentation feedstocks is currently impeded by the cumbersome process and high costs associated with biomass feedstocks and the conversion of biomass into sugars, including pretreatment and enzyme hydrolysis operations, which can account for up to 35% of total production costs. 7 , 8 Therefore, it is imperative to identify suitable energy crops and develop sustainable, cost-effective methods for pretreatment and hydrolysis to achieve maximum sugar conversion yields with minimal environmental impact. 9 Tobacco is a globally cultivated major commercial crop primarily used in the controversial smoking industry. It also serves as a model system in plant research, with its genome sequence published in 2014 and an extensive body of knowledge and engineering tools accumulated. 10 Genetic engineering has been utilized to enhance drought resistance, 11 , 12 increase leaf biomass production, 13 boost fermentable sugar content, and promote seed oil production in tobacco plants. 14 When grown for more biomass production, coppicing can be employed to stimulate re-sprouting and allow for multiple harvests. 15 Tobacco seed oil has been proposed for biodiesel production, while tobacco biomass has been tested for biogas production. 14 , 15 However, there have been limited studies examining the full potential of tobacco as an energy crop for its use as a renewable raw material in biorefineries for the production of chemicals and fuels. Energy crop plantations have traditionally occupied cropland, raising concerns regarding competition with food production, global food security, and environmental sustainability. 16 An alternative approach is to cultivate energy crops on barren lands, optimizing management strategies, utilizing genetic modifications, and developing new agricultural technologies. 17 , 18 Estimations of indirect carbon costs for corn grain ethanol range from 25 to 200 gCO 2 e/MJ, diminishing the environmental benefits of energy crops grown on arable land for biofuels. 19 Accordingly, energy crop biomass production on barren lands has been identified as a strategy that offers climate-related advantages. Furthermore, life cycle assessment (LCA) provides a method for evaluating the potential environmental impacts of a product system throughout its life cycle, 9 which helps in concluding the environmental benefits of biofuels derived from a specific material. Therefore, we aim to assess the environmental impact of tobacco cultivation and utilization, providing insights for policy-making related to sustainability that promotes its optimal use in bioenergy applications.",
"discussion": "Discussion Expanding the range of raw materials for next-generation biofuel production is crucial to enhance the effectiveness of biofuels and biochemicals. Initially, tobacco was identified as a potential candidate for biofuel due to its efficient oil biosynthesis mechanism in seeds. The mutagenized tobacco variety “Solaris” was subsequently found to possess an oil yield of 40%–60% of the seed’s dry weight, making it suitable for biodiesel production. 31 , 32 Researchers from the United States, Italy, and Poland recognized the potential of tobacco plants as an energy crop for biofuel production, 15 , 24 , 33 , 34 emphasizing their economic and financial viability. 35 Moreover, the decline in subsidies and tobacco consumption, along with significant waste in the tobacco industry, has highlighted the alternative utilization of tobacco plants for biofuel production. 36 The high leafiness of tobacco plants and the impact of maturity and drying methods on water-soluble components and sugar accumulation 37 support the superior performance of cured tobacco leaves in our study. Furthermore, tobacco stems have been evaluated as a sustainable energy source, 38 serving purposes such as biochar, 39 biomass raw materials, 40 energy storage material, 41 and botanical pesticides. 42 The direct utilization of tobacco leaves represents a revolutionary approach to traditional biomass production strategies. Considering this, our focus primarily lies in researching tobacco leaves as a medium and optimizing their utilization strategy for the production of bioproducts. In addition, sweet sorghum is also considered to be directly utilized as a medium by juicing and heating. However, the major challenges associated with biofuel production using sweet sorghum juice are short harvest period and fast sugar degradation during storage. 43 Additionally, sweet sorghum juice lacks a nitrogen source when used directly as a medium. However, the use of tobacco leaves as a medium can almost perfectly solve the above shortcomings in storage, transportation, and application. Nitrogen is indispensable for the growth of all living organisms, and its assimilation into various life-sustaining compounds has been extensively studied by microbiologists. 44 The nitrogen-rich tobacco makes it particularly attractive in nitrogen-rich compounds biosynthesis, such as guanidine (CH 5 N 3 ) and hydrazine. 45 Although our results indicated that nicotine in tobacco leaf medium had no effect on yeast growth or product production, with only a slight impact on E. coli , this can be addressed through nicotine removal or genetic engineering techniques. 46 , 47 Moreover, ongoing research on nicotine-free tobacco plants offers even greater potential for widespread tobacco utilization. In addition, the production of recombinant protein therapeutics, vaccines, and plasma products heavily relies on various expression systems ( E. coli , yeasts, mammalian cell culture, and insect cells) that are cultivated in media supplemented with animal-derived nitrogen components to support viability and productivity. These proteins are also commonly added as excipients and stabilizers in the final drug formulation. However, animal-derived raw materials carry a risk of viral contamination due to contact with viruses shed by animals. 48 , 49 Thus, the nutrient-rich tobacco, being a non-animal-derived component, can effectively mitigate the risk of virus transmission from animal sources, offering a more viable long-term solution. As a result, tobacco becomes an attractive alternative source for synthetic media in biorefineries. Economic feasibility is a crucial factor in the biomass-derived fuel controversy. 50 The major cost components in bioethanol production from lignocellulosic biomass are pretreatment and enzymatic hydrolysis steps. 51 , 52 , 53 Efficient pretreatment strategies can lead to substantial enzyme savings, as these processes are interconnected. Therefore, optimizing these two critical steps, which collectively account for approximately 70% of the total processing cost, presents significant challenges for the commercialization of bioethanol from second-generation feedstocks. 54 Additionally, previous studies have shown that tobacco leaves alone may not be profitable for biofuel production, while the project can become economically viable when focusing on high-value products, particularly high-value squalene. 35 This conclusion stems from the fact that the concept of using tobacco as a feedstock was in its early stages at that time, with technology primarily focused on extracting biofuels from tobacco leaves and seeds. Incorporating approaches that modulate metabolic pathways in microbial bioconversion to synthesize higher-value products can enhance the economic viability of biomass-derived biofuels, especially as fossil fuels are currently produced at significantly lower prices than biofuels. 55 By combining the production of higher-value products with biofuels through our simple and efficient tobacco utilization method, their economic viability can be increased. Bioenergy systems play a significant role in large-scale carbon dioxide removal (CDR), which is imperative for accomplishing climate goals by converting atmospheric CO 2 into carbohydrates. 56 Decades of research have led to a substantial knowledge base on enhancing CO 2 fixation and increasing dry matter productivity in tobacco, such as reducing the size of light-harvesting antenna in photosystems, 57 accelerating recovery from photoprotection, 58 and incorporating synthetic glycolate metabolic pathways into chloroplasts. Field experiments have validated that engineering photorespiratory pathways while inhibiting the native pathway can increase tobacco biomass by over 40%, benefiting from its ease of genetic transformation and robustness in the field. 13 Our analysis of actual global tobacco leaf production in 2019 reveals that even with tobacco leaves grown for high-quality cigarette products, the leaves harvested from barren lands have the potential to produce approximately 572 billion gallons of bioethanol, roughly 20 times the global ethanol production in 2019. While this framework analysis has limitations, such as the lack of real-world evidence for biomass production in barren areas, these outcomes have practical implications for the application and adoption of tobacco biofuels. The climate benefits of cellulosic biofuels have faced challenges regarding technological feasibility and carbon debt from indirect land-use changes, leading to calls for reduced support for large-scale deployment. 17 Nevertheless, it has been demonstrated that second-generation biofuels have greater potential to reduce GHG emissions (around 50%) compared to first-generation biofuels when land-use changes are not considered in LCAs. 51 In this context, we propose the use of barren soils for tobacco cultivation to address concerns about carbon losses from land-use change. Our LCA results indicate that tobacco-based ethanol already achieves a negative carbon footprint, thereby promoting further utilization of tobacco biomass. Enhancing the efficiency and sustainability of the tobacco crop requires an integrated approach involving agronomists, engineers, and farmers. Modern genetic approaches like CRISPR offer insights into improving the carbon fixation ability of crops and can be explored for reducing atmospheric CO 2 . 55 Restoring or enhancing the productivity of barren lands while utilizing them for biofuel cropping systems could contribute significantly to global energy and GHG mitigation goals, along with conservation benefits."
} | 3,434 |
38343971 | PMC10851232 | pmc | 4,204 | {
"abstract": "Triboelectric nanogenerators\n(TENGs) have been developed as promising\nenergy-harvesting devices to effectively convert mechanical energy\ninto electricity. TENGs use either organic or inorganic materials\nto initiate the triboelectrification process, followed by charge separation.\nIn this study, a high-performance composite-based triboelectric nanogenerator\n(CTENG) device was fabricated, comprising polydimethylsiloxane (PDMS)\nas a polymeric matrix, barium titanite (BTO) nanopowders as dielectric\nfillers, and graphene quantum dots (GQDs) as conductive media. The\nPDMS/BTO/GQD composite film was prepared with GQDs doped into the\nmixture of PDMS/BTO and mechanically stirred. The composition of the\nGQD varied from 0 to 40 wt %. The composite was spin-coated onto flexible\nITO on a PET sheet and dried in an oven at 80 °C for 24 h. The\noutput performance of TENGs is enhanced by the increased concentration\nof 30 wt % GQD, which is 2 times higher than nanocomposite films without\nGQD. The PDMS/BTO/G30 TENG film depicted an increase in open-circuit\nvoltage output ( V OC ), short-circuit current\noutput ( I SC ), and power density reaching\n∼310.0 V, ∼23.0 μA, and 1.6 W/m 2 , respectively.\nThe simple and scalable process for the PDMS/BTO/GQD TENGs would benefit\nas a sustainable energy-harvesting system in small electronic devices.",
"conclusion": "Conclusions The PDMS/BTO/GQD nanocomposite films are scientifically\ninvestigated\nand applied for energy-harvesting applications. The nanocomposites\nwere fabricated by the spin-coating method with different concentrations\nof GQD nanoparticles from 0 to 40 wt %. The output performance of\nTENGs is enhanced by the increased concentration of 30 wt % GQD which\nis 2 times higher than nanocomposite films without GQDs (PDMS/BTO\nonly). The best output performance is achieved by PDMS/BTO/G30, which\nproduces a voltage, current, and power density of ∼310.0 V,\n∼ 23.0 μA, and 1.6 W/m 2 , respectively. The\nstable and good electrical output power generated by the TENGs suggests\nthat the device has the potential for energy harvesting in nanoenergy\napplications.",
"introduction": "Introduction In the present Internet of Things (IoT)\nera, the demand for electricity\nin daily life is increasing at an unprecedented level with the popularization\nof portable electric and electronic devices. Normally, electricity\ncomes from conventional fossil fuels. However, the biggest concern\nis the fact that fossil fuels are limited resources and nonrenewable.\nThe use of fossil fuels also poses risks to the environment and also\naffects the carbon footprint. Therefore, remarkable efforts have been\ndeployed to replace conventional fossil fuel with renewable energy\nsources such as solar energy, wind energy, hydroelectric energy, and\nmany more alternative energy resources. Among various energy harvesters,\nthe mechanical energy harvester is a promising candidate as a new-generation\nenergy harvester capable of harvesting energy around the clock. Following\nthe first introduction of triboelectric nanogenerators (TENGs) in\n2012 by the Wang 1 group that successfully\nreported the use of piezoelectric nanogenerators (PENGs), there has\nbeen growing interest in developing alternative methods in scavenging\nfor the ambient mechanical energy from the environment to electricity.\nIn TENGs, two different triboelectric materials (either organic or\ninorganic) were rubbed together to initiate the triboelectrification\nprocess, followed by charge separation. TENGs are relatively cheaper\nas compared to PENGs and they are capable of producing higher output\npower and can be easily fabricated. 2 − 5 TENGs are excellent candidates for the potential\napplication in integrated energy-harvesting devices, which convert\nuntapped mechanical energies directly to electrical signals from various\nsources, such as wind flow, ocean waves, human motion, and even blood\nmovement inside human veins. 6 − 10 To date, various triboelectric polymeric materials have been used\nin TENGs, such as polymers [polyamide, polytetrafluoroethylene (PTFE),\npolydimethylsiloxane (PDMS), and polyvinylidene fluoride (PVDF)]. 11 − 13 In order to successfully apply TENGs as energy harvesters, these\nspecial types of generators must exhibit certain criteria such as\nbeing highly flexible, being able to maximize electrical output, and\nbeing robust in enduring high mechanical stress or strain. Even though\ntypical TENGs consisting of the above tribomaterials are preferred\ndue to the flexibility and good triboelectricity of the polymers,\nthe polymer-based TENGs are facing critical issues such as the inclination\nof the electron to recombine with positive charges induced on an electrode\nand low conductivity of the triboelectric polymers. PDMS is\none of the most negative triboelectric materials frequently\nused in TENGs given its ability to gain electrons while being highly\nflexible, highly electronegative, nontoxic, and biocompatible. Due\nto its simple preparation conditions and physical tunability, PDMS\nhas become a suitable choice for engineering properties in achieving\nhigh-performance TENGs. In order to increase the electronic characteristics\nof the PDMS-based TENG, incorporating high-dielectric inorganic materials\ninto a PDMS matrix could promote the relative permittivity and charge\ndensity of tribomaterials, which further boost the PDMS-based TENG\nelectrical output. 11 − 16 Various types of high-dielectric-constant inorganics materials such\nas TiO 2 , SrTiO 3 , BaTiO 3 (BTO), and\nZnSnO 3 17 − 19 were added into PDMS to improve the effectiveness\nof the composite film. Among all of the dielectric materials, BTO\nis one of the most appealing due to its high dielectric constant and\nferroelectric properties making it an ideal candidate for nanogenerators. 20 Graphene has been widely utilized in diverse\nelectrical devices\nand constructs. In a graphene monolayer, each carbon atom is covalently\nbonded with other nearby atoms to construct a honeycomb-like lattice.\nThe essential characteristics of graphene are its excellent thermal\nand electrical conductivity. Graphene has been reported to exhibit\nan electrostatic behavior and is able to store electrical charge when\nfriction is applied, thereby creating more opportunities for the potential\napplication of graphene in TENGs. 21 Graphene\ncan offer rich charge transfer pathways in TENGs and this leads to\nsignificant improvement in the output performance of the TENGs. 22 There are also reports on the use of dispersed\ngraphene quantum\ndots (GQDs) as fillers and as alternative candidates, besides the\ngraphene and graphene oxides, to increase the dielectric constant\nof polymer materials. 23 Unique optic, electronic,\nspintronic, and photoelectric properties induced by the quantum confinement\neffect and edge effect, including its fragments limited in size, or\ndomains, of a single-layer two-dimensional layered structure with\na large aspect ratio allow GQDs to form a large number of parallel\nmicrocapacitors within the polymer matrix. 24 − 26 In this\nstudy, a high-performance composite-based triboelectric\nnanogenerator (CTENG) device based on PDMS as a polymeric matrix with\nBTO nanoparticles as dielectric fillers and graphene as conductive\nmedia was fabricated. The three-phase nanocomposite is observed to\nexhibit a percolation system, in which BTO was uniformly and randomly\ndispersed in the polymeric matrix and surrounded by graphene, which\nformed discrete microcapacitor structures. The highest-voltage-, current-,\nand power-density-producing PDMS/BTO/GQD nanocomposite will be found\nto have the best output performance. The stable and good electrical\noutput power generated by the TENGs suggests that the device has the\npotential for energy harvesting in nanoenergy applications.",
"discussion": "Result and Discussion In order to\ndetermine the microstructure of PDMS/BTO/GQD nanocomposites,\nthe samples were analyzed by X-ray diffraction (XRD). As illustrated\nin Figure 2 , the broad\ndiffraction patterns at 2θ = 12° indicate that the PDMS\nwas amorphous, and the addition of BTO and GQDs did not change the\nbulk properties of the PDMS. Figure 2 XRD patterns of PDMS and PDMS/BTO. The XRD pattern of BTO confirms the cubic phase of BTO (JCPDS\nno.\n#892475). The peaks at 2θ = 22.12, 31.50, 38.75, 45.12, 50.23,\n55.80, 65.71, and 74.75° are indexed as (100), (110), (111),\n(200), (210), (211), (220), and (310) diffraction planes of the cubic\nphase, respectively. Meanwhile, a diffraction peak observed at 2θ\n= 26.2° (JCPDS no.—41-1487) corresponds to the signature\n(002) plane of the graphene quantum dot presented in the composite\nsample. 28 Figure 3 demonstrates\nthe characteristic peaks of different compositions of the PDMS/BTO/GQD\nnanocomposite films. There are no significant comparative shifts in\nthe diffraction position of the other polymer nanocomposite films.\nAll the peaks of PDMS and BTO nanoparticles were present and unchanged\nupon the addition of different compositions of GQDs. However, it can\nbe noticed that the diffraction peak is increased and broadens as\nmore GQDs are added to the nanocomposites. Figure 3 XRD patterns of (a) PDMS,\n(b) PDMS/BTO, (c) PDMS/BTO/G10, (d) PDMS/BTO/G20,\n(e) PDMS/BTO/G30, and (f) PDMS/BTO/G40. The presence of GQDs also has been further confirmed by Raman spectroscopy,\nthe Raman spectrum of a PDMS/BTO/GQD (PDMS/BTO/G4) that was excited\nusing a green laser (532 nm). The D and G peaks ( Figure 4 ) appeared in the band that\ncorresponded to the aromatic domain’s GQD. The peaks at 1350\ncm –1 can be related to the sp 3 orbital\nhybridized of C=C (carbon-to-carbon bonds), C=O (carbon-to-oxygen\nbonds), and COOH (hydroxyl group), respectively, while the peaks at\n1550 cm –1 correspond to the C–C due to the\nsp 2 carbon orbital. 29 Figure 4 Raman spectra\nof PDMS/BTO/G40. Scanning electron microscopy\nis one of the most effective methods\nand is useful to elucidate the surface morphology, surface topography,\nand composition of materials. FE-SEM characterization has been carried\nout to investigate the dispersion states of the BTO and GQD in the\nPDMS polymer matrix. Figure 5 a–f displays FE-SEM images of the neat PDMS film, PDMS/BTO,\nand PDMS/BTO/GQD nanocomposite films loaded with various compositions\nof GQD, respectively. Figure 5 FE-SEM images of the top surface of (a) PDMS, (b) PDMS/BTO,\n(c)\nPDMS/BTO/G10, (d) PDMS/BTO/G20, € PDMS/BTO/G30, and (f) PDMS/BTO/G40\nand close-up image of (g) PDMS/BTO/G30. The neat PDMS film exhibits a smooth surface, and the micrograph\nof the PDMS/BTO nanocomposite ( Figure 5 b) demonstrates the uniform distribution of BTO within\nthe polymer matrix, as indicated by the small circle in the image.\nUpon addition of the GQD ( Figure 5 c,f), the samples depict the numerous wormlike particles\ngathered to form a stacking structure. The surface modification is\nclearly demonstrated in which the nanocomposite film is seen to crumple,\nbut the BTO remains uniformly distributed, suggesting that the GQD\nforms a strong interaction with the host polymer PDMS. The nanocomposite\nfilms become more crumpled as the GQD concentration increases and\nthis crumpling effect provides an increased roughness in the systems,\nwhich could be useful for the application of TENGs. 30 In Figure 5 g, a close-up image of the PDMS/BTO/G30 is shown. The higher magnification\nmicrograph of PDMS/BTO/G30 with a 30% concentration of the GQD reveals\na clearly crumpled surface of the nanocomposites. The phenomena can\nbe explained as the increase of GQD content causing an increase in\nthe electrostatic and π–π interactions between\nthe GQD and PDMS. Furthermore, as the GQD concentration increases,\nthe uniform dispersion of GQDs can create a good network between the\npolymer matrix, BTO, and GQD, thereby improving the dielectric of\nthe PDMS/BTO/GQD samples. 31 The electrical\ncharacteristics of PDMS/BTO/GQD-based TENGs were\nevaluated by using a vertical contact-separation mode at a frequency\nof 1 Hz. All TENGs were subjected to an identical force and a 10 mm\nseparation distance between the composite film and field-effect transistor\n(FET) films. The working mechanism of the PDMS/BTO/GQD-based TENGs\nis described as follows: At the initial state as shown in Figure 6 a, the upper electrode\n(PET) was separated from the PDMS nanocomposite film, and there is\nno potential difference since there is no initial charge between the\nsubstrate and the electrodes. Figure 6 Working mechanism of the TENGs based on the\nvertical contact-separation\nmode at a frequency of 1 Hz. Then, the upper electrode was compressed by an external force and\nmoved into contact with the PDMS/BTO/GQD composite film. The static\ncharges were generated; refer to Figure 6 b. During the frictional process, PDMS/BTO/GQD\nand PET, which have high triboelectric negative and positive charges,\ntend to gain and lose electrons. The triboelectric effect causes positive\ncharges on the PET, while negative charges are formed on the PDMS/BTO/GQD\nsubstrate. The electron no longer flows to the external circuit as\nthe oppositely polarized inducted charges have reached equilibrium.\nUpon release of the external compression force, the electrostatic\nfield is developed. As the distance between the two layers increases\nas shown in Figure 6 c, the electric potential difference between the two layers is enhanced,\nthus allowing the electrons to flow and generate an instantaneous\ncurrent. Finally, the electron is redistributed as balance when the\ntriboelectric charges reach equilibrium again when the film is completely\nseparated; refer to Figure 6 d. The voltage output signal is produced repeatedly by contacting\nand separating the two electrodes using external force. The\nexperimental setup used for the measurements was adopted from\nthe current-to-voltage electronic circuit. 27 An Agilent N2873A oscilloscope was used for output voltage measurements.\nThe current output for triboelectric energy harvesters is very small\nin the range of μA, which is difficult to be measured directly.\nThe current-to-voltage converter with a conversion ratio of 100 mV/μA\nhas been assembled with the feedback resistor (100 kΩ), the\noutput voltage V o and input current I in is equal to the feedback resistor R f , ( V o / I in = − R f ) in the LMC6001\nop-amp inverting configured. 32 The voltage\nand current have been measured simultaneously on Channel-1 and Channel-2,\nrespectively. The instantaneous peak power of a TENG with an external\nresistance can be expressed as P = I 2 R where P is the instantaneous\npeak power, I is the current, and R is the resistance. Table 1 presents\nthe voltage readings according to the weight percentage of BTO from\n0 to 30%. It was found that the PDMS/20BTO combination recorded the\nhighest voltage. Table 1 Voltage Performance of the PDMS/BTO\nTENGs from 0 to 30 wt % of BTO samples design BTO (wt %) V OC (V) PDMS 0 73 PDMS/B5 5 86 PDMS/B10 10 95 PDMS/B15 15 122 PDMS/B20 20 156 PDMS/B25 25 132 PDMS/B30 30 128 Therefore, a weight percentage of 20% has been chosen\nfor the mixture\nwith GQDs. The PDMS/BTO/GQD composite film was prepared using the\nsame approach, with the GQD doped into the mixture of PDMS and BTO\nat various compositions between 0 and 40 wt %. Figure 7 a shows\nthe voltage output performance of the fabricated TENG devices with\ndifferent concentrations of PDMS/BTO/GQD composite films. The highest\nopen-circuit voltage ( V OC ) for the TENGs\nwith the PDMS/BTO/G30 film is ∼310.0 V. The V OC without the addition of GQDs can be shown in the sample\nPDMS/BTO as ∼156.0 V. Figure 7 (a) V OC , (b) I SC , and (c) their peak-to-peak value of the\nPDMS/BTO-based\nTENGs as a function of GQD content at pressing frequencies of 1 Hz. As shown in the measured short-circuit current\noutput ( I SC ) of the corresponding sample\nof Figure 7 b, the positive–negative\npeaks were repeated at pushing–separating points. Under 10 N /1 Hz of pushing force/frequency, the averaged output current\nwas obtained as ∼10.0 μA for the PDMS/BTO sample; the\ncurrent is observed to gradually increase when the GQD is added to\nthe matrix. The highest current output recorded, ∼23.0 μA,\nwas also found on the PDMS/BTO/G30 sample. The higher output\nperformance of the PDMS/BTO/GQD-based TENG can\nbe attributed to the following reasons: BTO is a good piezoelectric\nmaterial and when the piezoelectric is added into the triboelectric\nsystem, the piezoelectric effects can significantly enhance the performance\nof the TENG. 33 When a mechanical force\nis applied, the triboelectric material PDMS, which contains piezoelectric\nBTO, undergoes deformation, leading to an immediate demonstration\nof piezoelectricity. The piezoelectric effect of BTO produced the\nsupplementary surface charges, thus reinforcing the triboelectric\ncharge during simultaneous contact electrification and ultimately\nboosting the voltage outputs. The performance of TENGs is closely\nrelated to the dielectric properties of the triboelectric materials.\nThe dielectric improvement of the triboelectric materials can improve\nthe charge-trapping capability which enhances the surface charge density\nof the PDMS matrix and results in high output performance of the TENGs. 34 , 35 The TENGs’ structure can be treated as an equivalent electric\ncircuit model, which is simplified as three capacitors connected in\nseries. 36 The triboelectric potential (V)\nfor the TENG can be described as follows 1 where σ and Q are the surface charge\ndensity and the induced charges distributed on the dielectric surfaces\nand the electrode surfaces. While d and ε represent\nthe thickness and permittivity of the triboelectric materials, S is the electrode area, X ( t ) is the gap distance between two triboelectric materials surfaces,\nand ε 0 is the vacuum permittivity. 36 The surface charge density (σ), induced\ncharges ( Q ), and permittivity of the dielectric materials\nare the key elements for the output of the TENGs. Under a fixed surface\ncharge density (σ), the output of the TENGs can be enhanced\nby increasing the permittivity of the triboelectric materials. The\nequation shows that the permittivity increase of both triboelectric\nmaterials can enhance the electric output of the TENG. The presence\nof the GQD particles in the PDMS matrix is expected to provide more\narea for interfacial polarization, which can considerably raise the\npermittivity of the PDMS nanocomposites for static charge storage.\nWhen the GQD is added into the PDMS nanocomposite system, the effective\npermittivity of the PDMS nanocomposites also increases and greatly\nincreases the charge density on the PDMS surface. The study also showed\nthat the GQD played a key role in the increase of dielectric constant\nand reduced dielectric loss. 26 The ternary\nstructure promised the possibility of fabricating polymer composite\nmaterial with excellent flexibility, high dielectric constant, and\nlow dielectric loss by incorporating ceramic fillers and GQDs in the\npolymer matrix. Thus, increasing the number of GQDs will improve the\ncapacitance of the device by simply increasing the energy storage\ncapacity to generate a high electrical output. It can be described\nthat the addition of graphene in the PDMS matrix\nhelps the electrons attracted from the PDMS matrix through the friction\nprocess to either be stored in the discrete, quantized levels of these\nnanosized graphene particles or trapped in the GQD dielectric. This\nperformance improvement is attributed to the residual charges accumulated\nby graphene, increasing the triboelectric current. It can also be\nnoticed from the results shown in Figure 7 a,b that the electrical output performance\nof TENGs for PDMS/BTO/G40 was slightly reduced. The performance decreases\nare probably contributed by the excessive presence of GQDs on the\nPDMS surface, which reduces the effective contact surface area. 37 When the GQDs are added beyond the optimum concentration,\nit also could cause the PDMS/BTO/GQD nanocomposites to agglomerate,\ncreating an increase in the leakage current and a decreased dielectric\nproperty of the PDMS/BTO/GQD; this phenomenon can be described by\npercolation theory. 38 Furthermore, PDMS\n(high tribonegativity in the triboseries table) is a much better triboelectrification\nmaterial than BTO and GQD, and the decrease of the PDMS matrix surface\narea can also lead to the decrease in generated static charge. For\nvalidation of the theory, the use of graphene as triboelectric materials\nin TENGs has been compared and tabulated in Table 2 . 11 A peak-to-peak\nvalue in Figure 7 c\nconfirmed that the PDMS/BTO/G30 sample shows the highest V OC and I SC values. As a result,\nthe PDMS/BTO/G30 sample was then chosen for further investigation\nby connecting these samples to an external load with a resistance\nrange of 1 kΩ–30 MΩ. Table 2 Comparison\nof the Graphene-Based TENGs\nin Vertical Contact-Separation Mode negative\ntriboelectric materials positive\ntriboelectric materials device dimension\n(cm) voltage (V) current (μA) power density\n(Wm 2– ) PDMS/3D bilayer graphene/carbon cloth (PDMS/G/CC) 22 PET 3.0 × 3.0 70 0.0065 PDMS/graphene 31 PET 2.2 × 2.2 270.2 1-Layer graphene 39 PET 5 Graphene/EVA/PET film 40 PDMS 3.0 × 4.0 22 PDMS/GO/SDS 41 PEN 2.0 × 2.5 438 PVDF/GQD 42 Al 6.0 × 6.0 ∼120 ∼3 Al-doped ZnO/GO 43 PI 2.0 × 2.0 105 PDMS/graphene-Ag nanowires 44 PDMS 10.0 × 10.0 GN/PTFE 45 Al 3.0 × 3.0 96 3.66 3.9 PI/PI:rGO/PI 46 Al 1.5 × 2.5 190 6.3 As shown in Figure 8 , when a load resistance is applied, the\ncurrent reduces, because\nthe charge transfer mechanism inside the resistance range is identical\nto the short-circuit condition. The output current approaches zero\nby further increasing the resistance beyond 1 MΩ. By varying\nthe load resistance from 1 kΩ to 5 MΩ, the value of power\ndensity rises dramatically from 0.102 to 1.6 W/m 2 . However,\nas the load resistance exceeds 5 MΩ, the power density is reduced\nand reaches a value of 0.86 W/m 2 at 30 MΩ. Figure 8 Maximum output\ncurrent and power as a function of the external\nload resistance. Therefore, it is believed\nthat there certainly exists an optimal\nload resistance where the maximum output power is achieved. For validation\nof the performance, a comparison of the TENGs based on graphene is\nsummarized in Table 2 ."
} | 5,565 |
35330239 | PMC8953515 | pmc | 4,207 | {
"abstract": "Fungal endophytes have been extensively found in most terrestrial plants. This type of plant–microorganism symbiosis generates many benefits for plant growth by promoting nutrient availability, uptake, and resistance to environmental disease or stress. Recent studies have reported that fungal endophytes have a potential impact on plant litter decomposition, but the mechanisms behind its effect are not well understood. We proposed a hypothesis that the impacts of fungal endophytes on litter decomposition are not only due to a shift in the symbiont-induced litter quality but a shift in soil microenvironment. To test this hypothesis, we set-up a field trial by planting three locally dominant grass species (wild barley, drunken horse grass, and perennial ryegrass) with Epichloë endophyte-infected (E + ) and -free (E − ) status, respectively. The aboveground litter and bulk soil from each plant species were collected. The litter quality and the soil biotic and abiotic parameters were analyzed to identify their changes across E + and E − status and plant species. While Epichloë endophyte status mainly caused a significant shift in soil microenvironment, plant species had a dominant effect on litter quality. Available nitrogen (N) and phosphorus (P) as well as soil organic carbon and microbial biomass in most soils with planting E + plants increased by 17.19%, 14.28%, 23.82%, and 11.54%, respectively, in comparison to soils with planting E − plants. Our results confirm that fungal endophytes have more of an influence on the soil microenvironment than the aboveground litter quality, providing a partial explanation of the home-field advantage of litter decomposition.",
"conclusion": "5. Conclusions In conclusion, our findings verified the hypothesis that Epichloë endophytes did affect both the initial litter quality and the soil environment. Importantly, we showed that endophyte status had more host-dependent effects on soil biotic and abiotic factors compared with their effects on host litter properties. In contrast, plant species had only dominant effects on litter properties. The endophyte-induced shifts in soil nutrient availability and microbial activities could lead to a significant promotion of litter decomposition and thus assist our understanding about the home-field advantage of litter decomposition. Our findings suggest a new research direction in the future that could focus on performing studies involved in the impacts of key ecological processes and ecosystem functions induced by fungal endophytes.",
"introduction": "1. Introduction Plant–microbe symbioses exist widely in the grassland ecosystem. The symbiosis can exert great effects on both the growth and the physiology of host plants and on the microenvironment [ 1 , 2 ]. Most studies about microbial symbioses have focused on the mycorrhizal fungi and nitrogen-fixing bacteria due to their well-known beneficial effects on host plants [ 3 , 4 , 5 , 6 ]. However, the functional significance of other microbial symbioses, such as fungal endophytes, is much less understood to date [ 7 , 8 ]. Recent studies have shown that endophytic fungi play an essential role in enhancing the resistance and adaptability of host plants in grassland communities [ 9 , 10 , 11 , 12 ], but their potential impact on the host litter components and the soil environment across plant species has been largely overlooked [ 13 ]. Fungal endophytes are defined as plant-associated fungi that colonize, and live symbiotically within, plant tissues (e.g., leaves and stems) during a specific phase of their life. Generally, they are not harmful to their hosts when taking up residence in host organisms [ 8 , 14 , 15 ]. Fungal endophytes have been detected in approximately 30% of grass species [ 16 ]. They receive nutrients and protection from their host plants, and transmit from generation to generation by vertical transmission through host plant seeds [ 17 ]. In return, fungal endophytes protect their host plants from pathogens by producing secondary metabolites [ 18 , 19 ] and cell wall-degrading enzymes [ 20 ], or by inducing systemic resistance [ 21 ]. Moreover, they are capable of protecting their hosts against several environmental stresses [ 22 ] such as drought [ 23 ], salinity [ 24 ], nutrient depletion [ 25 ], flooding [ 26 ], and thermal stress [ 8 ]. As such, fungal endophytes increase their host’s fitness and they are likely to follow changes in their host’s morphological and physiological traits that are associated with nutrient acquisition, including a structural modification of plant tissues [ 27 ]. This may thus induce a shift in litter components or root exudates of host plants [ 28 , 29 ]. Epichloë is a typical genus affiliated with ascomycete fungi that commonly forms an endophytic symbiosis with grasses [ 30 , 31 ]. The symbiotic interaction between Epichloë endophytes and their hosts has been shown to affect many key ecosystem processes in different ways such as litter decomposition and soil nutrient cycling [ 32 , 33 , 34 ]. For example, Epichloë endophytes are able to induce a shift in chemical properties of aboveground host litter; and, consequently, they have an effect on litter decomposition [ 35 , 36 ]. The soil microenvironment tends to also be different between Epichloë endophyte-infected (E + ) and -free (E − ) plants due to host-induced root exudates, which thus strongly influence microbial decomposer communities by altering substrate quality and quantity [ 37 ]. Despite an increasing awareness of the fungal endophytes role in decomposition, few studies have been conducted to identify the mechanisms that fungal endophytes affect in litter decomposition [ 38 ]. In this study, we collected the aboveground litter and rhizosphere soils of Lolium perenne L. (perennial ryegrass), Hordeum brevisubulatum (Trin.) Link (wild barley), and Achnatherum inebrians (Hance) Keng (drunken horse grass), which have been demonstrated to form symbiosis with the Epichloë endophytes [ 39 , 40 , 41 ]. We hypothesized that foliar endophytic fungi would change the initial quality of the host litter and the soil microenvironment and that such an effect would vary across different host plant species. We aim to explore in the field (1) the shifts in litter quality and soil physicochemical and microbial properties across E + and E − status and plant species and (2) the differences in the effects of endophyte status and plant species on litter and soil properties.",
"discussion": "4. Discussion The formation of plant–endophyte symbiosis generally reflects a mutualistic strategy to cope with environmental stress for symbionts. The plant–endophyte symbiotic interactions help to promote the coevolution of hosts and fungal endophytes [ 55 , 56 ], maintenance of biodiversity and plant and soil health [ 57 , 58 ]. In this study, we attempted to link the fungal endophyte status and its host-dependent effects to litter decomposition by endophyte-induced changes in litter and soil properties. We showed that the presence of the Epichloë in host plants increased the contents of soil available nutrients (SOC, AN, and NN). However, host specificity has a larger impact on litter quality than the effect of endophytic fungi. The findings provided insights into how the foliar Epichloë fungal endophyte symbiotic with wild barley, drunken horse grass, and perennial ryegrass affected the initial quality of litter in the host plant and the microenvironmental conditions of decomposition. Most studies suggest that fungal endophyte–host plant interactions are mutualistic [ 59 , 60 ], but the interactions between the host–plant species and endophyte status are variable, ranging from positive to negative effects on litter decomposition (including litter quality and soil properties) [ 61 , 62 ]. The genetic factors of the plant species, endophyte status, and environmental factors can modify the nature of the symbiosis [ 63 , 64 ]. In this study, the effects of three host plants on litter quality and soil properties were inconsistent between E + and E − status. This is probably because the mutualistic symbioses depend not only on the presence of the endophyte but also on various abiotic factors and the network of species that interact with the host plant directly or indirectly [ 65 , 66 ]. The surveyed grass species and endophyte could thus play a decisive role in determining the nature of the grass–endophyte symbiosis. We provided evidence for the effect of fungal endophyte on aboveground litter quality because of the significant differences observed in some litter chemical components between E + and E − status. A distinct increase in ADF and ADL content but a decrease in cell soluble content was generally found in our study. This finding is consistent with several previous reports showing that ADF or ADL increased within internal plant leaf tissues when plants are infected by fungal endophytes [ 67 , 68 ]. We cannot arbitrarily make a conclusion that plants infected by fungal endophytes may increase or decrease these chemical components because different plant species or species with different genotypes may respond completely differently to endophyte status. However, this endophyte-induced shift in host organisms may indeed indicate a response strategy of plant physiology in certain environmental conditions [ 69 ]. It is worth pointing out that aboveground litter properties are inclined to be mostly affected by plant species [ 70 ]. This is actually reasonable because compared with the endophyte-induced alternation of hosts organisms, the content of various chemical components in live and dead plant tissues are highly different among plant species [ 71 ]. Through this field experiment we surprisingly found that fungal endophytes had strong influences on most examined soil physicochemical parameters, particularly involved in soil nutrients such as SOC, AN, NN, and AP content, etc. This interesting finding provided an additional clue to link plants with different endophyte statuses to altered soil microenvironments. It is though difficult to identify direct or indirect relationships between them based on our current data set, such a correlation may suggest some potential processes. For instance, studies have shown that the quality and the quantity of root exudates of plants can experience great changes when they are infected by fungal endophytes [ 72 , 73 ], which can consequently lead to a shift in microbe-mediated soil nutrient pools. Alternatively, this linkage possibly resulted from interactions between the plant–soil microbiome for nutrient competence and transmissions [ 74 ]. Increasing the soil available carbon (C), nitrogen (N), and phosphorus (P) content in E + plots across three plant species also suggests a beneficial effect of Epichloë endophytes on the host plants, in line with most previous reports [ 75 ]. In the long term, fungal endophytes may thus contribute greatly to plant and soil health in ecosystems. In contrast to endophytes status, plant species had very small and insignificant impacts on examined soil properties. This is not in accord with most studies conducted in grassland ecosystems [ 76 , 77 ]. The inconsistency may relate to similar physiological responses from selected plant species. In general, litter decomposition is affected by two major factors including initial litter quality and the decomposition environment. Therefore, based on the findings we mentioned above, Epichloë endophytes may have a positive effect on litter decomposition processes via altering initial host litter and soil biotic and abiotic properties. Our data provided supportive evidence such as increased litter N and P contents and decreased soil C/N ratio, as well as significant positive correlations between increased soil nutrient and microbial biomass in E + plots. Firstly, higher N and P concentrations have commonly indicated faster decomposition rates [ 78 ]. For example, previous studies have shown that the primary phase of litter decomposition was constantly positively correlated with the initial litter N or P concentration [ 79 ]. Secondly, litter N and P content, as primary energy resources for soil microorganisms, are often positively correlated with microbial activities in the decomposition process [ 80 , 81 ]. Hence, increased initial litter N and P concentration with E + status probably suggest a beneficial effect on litter decomposition. Furthermore, the decreased C/N ratio and the increased microbial biomass resulting from increased nutrients in soils with planting E + plants across three selected grass species provides further evidence to support this point as a number of studies have indicated their positive effect on promoting litter decomposition [ 82 , 83 ]."
} | 3,197 |
18676212 | null | s2 | 4,208 | {
"abstract": "We cross-linked scaffolds of electrospun collagen to varying degrees with glutaraldehyde using an ethanol-based solvent system and subsequently defined how the percentage of cross-linking impacts bulk and microscale material properties and fiber structure. At hydration, electrospun fibers underwent coiling; the extent of coiling was proportional to the percentage of cross-linking introduced into the samples and was largely suppressed as cross-linking approached saturation. These data suggest that electrospun collagen fibers are not deposited in a minimal energy state; fiber coiling may reflect a molecular reorganization. This result has functional/structural implications for protein-based electrospun scaffolds. Changes in fiber topology that develop during post-electrospinning processing may alter monomer organization, mask or unmask receptor binding sites, and/or change the biological properties of these nanomaterials. Hydrated scaffolds were mounted into a custom stretching device installed on a microscope stage and photographed after incremental changes in strain. Changes in fiber alignment were measured using the two-dimensional fast Fourier transform method. Fibers in all scaffolds underwent alignment in response to strain; however, the rate and extent of alignment that could be achieved varied as a function of cross-linking. We propose four distinct modes of scaffold response to strain: fiber uncoiling, fiber reorientation, fiber elongation and interfiber sliding. We conclude that bulk material properties and local microscale architecture must be simultaneously considered to optimize the performance of electrospun scaffolds."
} | 414 |
35479772 | PMC9036824 | pmc | 4,209 | {
"abstract": "Poly(ethylene terephthalate) (PET), known for its clarity, food safety, toughness, and barrier properties, is a preferred polymer for rigid packaging applications. PET is also one of the most recycled polymers worldwide. In light of climate change, significant efforts are underway to improve the carbon footprint of PET by synthesizing it from bio-based feedstocks. Often times, specific applications demand PET to be copolymerized with other monomers. This work focuses on copolymerization of PET with a bio-based co-monomer, 2,5-furandicarboxylic acid (FDCA) to produce the copolyester (PETF). We report the multifunction of FDCA to influence the esterification reaction kinetics and the depolymerization kinetics ( via alkaline hydrolysis) of the copolyester PETF. NMR spectroscopy and titrimetric studies revealed that copolymerization of PET with different levels of FDCA improved the esterification reaction kinetics by enhancing the solubility of monomers. During the alkaline hydrolysis, the presence of FDCA units in the backbone almost doubled the PET conversion and monomer yield. Based on these findings, it is demonstrated that the FDCA facilitates the esterification, as well as depolymerization of PET, and potentially enables reduction of reaction temperatures or shortened reaction times to improve the carbon footprint of the PET synthesis and depolymerization process.",
"conclusion": "Conclusions The effect of copolymerization with FDCA on the dissolution and esterification of TPA with EG was studied in detail in this work. End group conversion analysis based on NMR revealed that the co-esterification reactions occurred ‘in series’ rather than ‘in parallel’ as FDCA reacted with EG almost instantaneously to form FDCA-rich oligomers followed by esterification of TPA with EG. The presence of FDCA-rich oligomers promoted the dissolution of TPA in the reaction mixture and improved the TPA esterification reaction kinetics for PETF copolyester samples compared to PET homopolyester. The carboxyl end group conversions obtained from the NMR were confirmed by titrimetry validating the method used. MALDI-MS results showed that the copolymerization did not affect the degree of polymerization, however, the number average molecular weight increased marginally at shorter reaction times by copolymerization with FDCA. This increase was attributed to the improved kinetics at shorter times as shown by the NMR results. FDCA was predicted to be more acidic than TPA based on the calculated values of p K a . Since esterification is an acid-catalyzed reaction, the lower p K a and thus, the higher acidity of FDCA could also contribute to the enhancement of kinetics in addition to the co-solvent effect of FDCA-rich oligomers. As demonstrated in the ESI, † the higher acidity of FDCA also resulted in increased DEG production, a side product of the esterification step. Based on the improved esterification kinetics, it is proposed that the esterification step of PETF copolyesters can be performed at lower temperatures or times and reduce the energy consumption during the synthesis step. Due to the reversible nature of the condensation reactions, the effect of copolymerization with FDCA on depolymerization kinetics of PET was also investigated through a proof-of concept alkaline hydrolysis reaction. PETF20 copolymers showed almost double equilibrium conversion and monomer yield compared to PET homopolymer. A more comprehensive study on depolymerization of PETF copolymers at industrially relevant temperatures and recovery of FDCA from the diacid mixture will be published in a subsequent article.",
"introduction": "Introduction In the new age of circular economy and heightened awareness of climate change, polyethylene terephthalate (PET) has gained greater acceptance as a circular packaging polymer due to its excellent properties and recyclability. 1–6 Industrially, PET is synthesized either by transesterification of dimethyl terephthalate (DMT) with ethylene glycol (EG) (DMT process) or direct esterification of terephthalic acid (TPA) with EG (TPA process). 7 With advancement in processes to obtain purified TPA, the capacity of PET produced from the direct esterification of TPA and EG has increased significantly in the last two decades. The esterification step of the TPA process is a dissolution limited process due to the extremely low solubility of TPA in EG. 7,8 Higher reaction temperatures and pressures are required for the esterification step to dissolve the TPA in EG. Commercially available grades of PET used for packaging application generally contain small quantities of comonomers to modulate properties required for injection and stretch blow molding of PET. 9 In the production of PET via TPA process, comonomers like isophthalic acid (IPA) are introduced in the free diacid form along with TPA. During the copolymerization, the comonomer diacid may compete with TPA for the esterification reaction with EG and may affect the dissolution and reaction kinetics, especially at higher molar contents. While detailed studies on the role of solubility in the esterification kinetics of PET have been reported, 10–12 to the authors' knowledge, no published study on the effect of copolymerization on the esterification step of PET synthesis is available. In the recent past, significant research effort has been devoted to develop bio-based alternatives to PET. 13–17 Poly(ethylene 2,5-furandicarboxylate) (PEF), which can be produced from bio-based 2,5-furandicarboxylic acid (FDCA) and EG, has gained significant academic and industrial interest due to its superior properties and structural similarity to PET. 18–27 Due to limited availability, high costs, and limited mechanical recycling compatibility of PEF with PET stream, copolymerization or blending of PET with PEF is being considered as an effective approach to avail the enhancements of PEF without affecting the cost or recyclability of the final packaging. 27–30 In our previous work, we reported that FDCA has much higher solubility in EG compared to TPA. 31 This difference in the solubility may play a role during the esterification step for synthesis of copolyesters of FDCA and TPA with EG (PETF copolyesters). The primary focus of this work was to study the kinetics of co-esterification of TPA and FDCA with EG. The composition range of FDCA in the copolyesters was restricted to 20 mole percent due to the practical limitations associated with the cost of FDCA and recyclability of the copolyesters. Esterification kinetics were performed at two different temperatures of 250 °C and 225 °C. The temperature of 250 °C was chosen since the esterification step of PET synthesis is typically done at 250 °C commercially. 7 The temperature of 225 °C was chosen to evaluate the possibility to perform the esterification of PETF copolyesters at milder conditions than those typically used for PET commercially. Since the advantage of higher solubility of FDCA in EG can play a crucial role only in the first step of the polymerization, this study was restricted to the analysis of the reaction conditions for esterification step of PET production. A previously developed NMR method 32 was modified to track the end group conversion of hydroxyl and carboxyl end groups (for both diacids, TPA and FDCA) during the reaction. The end group conversion was also confirmed by titrimetry to validate the NMR method. Lastly, proof-of-concept alkaline hydrolysis experiments were carried out with PET and PETF20 (copolyester with 20 mole% FDCA) to investigate the potential effect of the presence of FDCA on reaction kinetics for PET depolymerization. In summary, this work is focused on developing a mechanistic understanding of effect of FDCA on esterification and depolymerization kinetics of PET.",
"discussion": "Results and discussion As described in the Introduction section, the TPA process is a dissolution limited process due to limited solubility of terephthalic acid (TPA) in ethylene glycol (EG). 7,8 Higher temperatures and pressures are required to dissolve the TPA in EG. In the case of synthesis of copolymers of PET via TPA process, the co-monomers like isophthalic acid are introduced in the diacid form for compatibility with the process. During the copolymerization, the co-monomer diacids are expected to compete with TPA for the esterification reaction with EG. Fig. 1 depicts a detailed reaction scheme for co-esterification of 2,5-furandicarboxylic acid (FDCA) and TPA with EG. Since this is a dissolution limited process, 7,8 the difference in the solubilities between FDCA and TPA can potentially affect the esterification kinetics and copolymer microstructure. In our previous work, we reported that FDCA has much higher solubility in EG compared to TPA. 31 Higher solubility of FDCA in EG could potentially translate into faster conversion of FDCA to bis-hydroxyethyl 2,5-furandicarboxylate (BHEF) and oligomers. During PET synthesis, oligomers of PET (bis-hydroxyethyl terephthalate or BHET) are often added to improve solubility of TPA in EG solution. 7,8 The BHEF and FDCA-rich oligomers formed during the co-polymerization could provide an improved solubility effect similar to BHET. Fig. 1 Series of chemical reactions involved during the co-esterification of diacid X (TPA and FDCA) with EG; MHEF = monohydroxyethyl 2,5-furandicarboxylate, MHET = monohydroxyethyl terephthalate, BHET = bishydroxyethyl terephthalate, BHEF = bishydroxyethyl 2,5-furandicarboxylate, PETF = poly(ethylene terephthalate- co -2,5-furandicarboxylate). To evaluate the effect of FDCA on dissolution and esterification kinetics of PET, direct esterification of copolymers of PET with FDCA was performed at 10 and 20 mole% of FDCA. As mentioned in the Experimental section, the protocol for industrial scale synthesis of PET was used for the esterification reactions for greater applicability of this work. The direct esterification was carried out at a commercially applicable temperature of 250 °C and a milder temperature of 225 °C to understand the effect of temperature on the kinetics. For simplicity, the copolyesters of PET with 10 and 20 mole% of FDCA are referred as PETF10 and PETF20, respectively. Note that the results and discussion are limited to the oligomers obtained during the esterification step. End group conversion by NMR \n 1 H and 13 C NMR spectroscopy was used to track the hydroxyl and carboxyl end group conversion during the esterification of TPA and FDCA with EG. The details of the method are described in the ESI. † Fig. 2 shows the hydroxyl end group conversion ( X OH ) during the esterification of PETF co-polyesters at two esterification temperatures. As seen from Fig. 2(b) , the reaction kinetics were much slower at 225 °C and the reaction had to be run for a longer time to achieve equilibrium conversions. At commercial synthesis conditions of 250 °C, equilibrium conversion was reached within one hour of reaction time for the co-polyesters. Please note that the esterification reaction conditions, and the reactor design were optimized to effectively remove the water generated during the esterification reaction without losing the EG in the condensate. Based on this assumption of no loss of EG, the equilibrium values of X OH were close to the theoretical conversion of 0.66 expected with 1.5 : 1 excess of hydroxyl end groups. Fig. 2 Hydroxyl end group conversion determined by 1 H NMR for PET ( ),PETF10 (-) and PETF20 ( ) for direct esterification performed at (a) 250 °C and (b) 225 °C. The hydroxyl end group conversion increased significantly for the PETF co-polyesters compared to PET at 250 °C, especially at low reaction times. On the other hand, at 225 °C, the increase in X OH following copolymerization was much less evident possibly due to the reduced solubility of TPA in EG and slower reaction kinetics compared to 250 °C. The X OH values at both temperatures for the PETF10 and PETF20 samples at zero reaction time were greater than 0 indicating the onset of the reaction during the ramping step. However, the PET sample did not show any conversion of hydroxyl groups during the ramping step. This observation indicated that the FDCA starts reacting with the EG at temperatures much lower than the TPA confirming the results published in our previous work. 31 The hydroxyl end group conversion was also employed to track the production of diethylene glycol, a known side reaction during the esterification step with a detailed discussion in the ESI. † Acid end group conversion was determined for FDCA and TPA independently using the 13 C NMR spectra as discussed in the ESI. † Total carboxyl end group conversion ( X COOH,total ) was calculated from the FDCA ( X COOH,FDCA ) and TPA ( X COOH,TPA ) end group conversions using eqn (4) . 4 X COOH,total = x × X COOH,FDCA + (1 − x ) × X COOH,TPA where x is mole fraction of FDCA in the polyester. As shown in Fig. 3 , X COOH,total followed a similar trend to that of X OH . Total acid end group conversion increased for the PETF copolyesters compared to PET. As was seen for X OH , the increase was significant especially at low reaction times. At 250 °C, the X COOH,total values converged at higher times ( Fig. 3(a) ) to a value of 0.87. However, this convergence was not observed in the time scale studied at 225 °C ( Fig. 3(b) ) indicating the slower kinetics at 225 °C. Fig. 3 Total carboxyl end group conversion determined by 13 C NMR for PET ( ), PETF10 (-) and PETF20 ( ) for direct esterification performed at (a) 250 and (b) 225 °C. \n Fig. 4(a) and (b) show the conversion of FDCA end groups at 250 and 225 °C respectively. The FDCA end groups reacted almost instantaneously at both reaction temperatures. Interestingly, almost half of the end groups were esterified during the ramping step ( X COOH,FDCA ∼ 0.5 at t = 0). As reported in our previous work, FDCA exhibits an order of magnitude higher solubility in EG compared to TPA at temperatures higher than 180 °C. 31 Additionally, in the case of copolymers, FDCA was dissolved in a very large molar excess of EG (15 : 1 in case of 10 mole% FDCA since total molar ratio of EG: diacid was set to 1.5 : 1). High solubility and molar excess of EG to FDCA resulted in an instantaneous dissolution and esterification of FDCA end groups. This rapid esterification of FDCA end groups was primarily responsible for the increase in the X OH and X COOH,total at low times. Fig. 4 FDCA end group conversion determined by 13 C NMR for PETF10 (-) and PETF20 ( ) for direct esterification performed at (a) 250 °C and (b) 225 °C. As shown in Fig. 1 , esterification of TPA and FDCA with EG could be considered as competing parallel reactions. However, within 15 minutes of the reaction time at 250 and 225 °C, most of the FDCA was esterified ( X COOH,FDCA ∼ 0.9). On the other hand, most of the TPA was unreacted after 15 minutes ( Fig. 5 ). Hence, due to the instantaneous esterification of FDCA, it is proposed that the reactions occurred in series rather than in parallel. Additionally, as shown in Fig. 5(a) and (b) , TPA end group conversions increased for PETF10 and PETF20 samples compared to PET. Fig. 5 TPA end group conversion determined by 13 C NMR for PET ( ), PETF10 (-) and PETF20 ( ) for direct esterification performed at (a) 250 °C and (b) 225 °C. At 250 °C, higher X COOH,TPA was obtained in the case of PETF10 and PETF20 over the full reaction time. However, at 225 °C, the improvement in X COOH,TPA for PETF samples occurred after a specific delay time as shown in Fig. 5(b) with arrows. This delay time observed at 225 °C was shorter for PETF20 (30 minutes) compared to PETF10 (180 minutes). Based on this observation, it is hypothesized that a certain concentration of esterified FDCA-rich oligomers in the reaction media was necessary to facilitate the conversion of TPA at 225 °C. This concentration was achieved at shorter times for PETF20 compared to PETF10 due to the higher molar ratio of FDCA in the feed for PETF20, and hence higher concentration of BHEF and FDCA-rich oligomers. In summary, hydroxyl and total carboxyl end group conversions increased with the presence of FDCA in the reaction media. The FDCA carboxyl end groups were almost instantaneously converted to the esterified products. These esterified FDCA-rich oligomers accelerated the conversion of TPA carboxyl end group in PETF copolyesters compared to PET. At 250 °C, X COOH,TPA of 0.84 was achieved for PET sample after 60 minutes of esterification reaction. A similar value of conversion was achieved in 45 minutes for PETF20 reducing the reaction time by 15 minutes or 25%. Esterification performed at 225 °C showed significantly slower reaction kinetics compared to 250 °C. Surprisingly, even at 225 °C, almost all of the FDCA was esterified within the first 15 minutes of reaction. TPA carboxyl end group conversions at 225 °C showed a delay time before the improvement due to esterified FDCA products was observed. Composition and degree of randomness by NMR \n 1 H NMR was also employed to investigate the composition and the degree of randomness of the growing chains as a function of esterification time and temperature. The oxyethylene proton peak corresponding to the in-chain EG unit showed peak splitting depending on the acid units surrounding it. Based on the area under the peaks, the mole fractions and degree of randomness of PEF and PET blocks was calculated (as explained in the ESI † ) and summarized in Table 1 . Composition or mole% of furan in PETF copolyesters and degree of randomness values (in parenthesis) as a function of esterification time calculated using the 1 H NMR spectroscopy Time (min) 250 °C 225 °C PETF10 PETF20 PETF10 PETF20 0 49 ± 01 (1.04) 69 ± 01 (0.56) 66 ± 04 (0.83) 85 ± 05 (1.23) 5 22 ± 07 (1.03) 47 ± 04 (0.85) — — 10 19 ± 01 (1.50) 33 ± 01 (1.00) — — 15 15 ± 03 (1.53) 26 ± 01 (0.99) 35 ± 03 (1.37) 56 ± 03 (0.86) 30 11 ± 02 (1.26) 25 ± 03 (1.00) 24 ± 01 (1.10) 41 ± 01 (1.01) 45 — 21 ± 02 (1.33) — — 60 — 26 ± 02 (1.13) 20 ± 02 (1.22) 39 ± 01 (0.95) 120 — 22 ± 04 (1.10) 18 ± 01 (1.12) 31 ± 03 (0.95) 180 — — 19 ± 01 (1.24) 24 ± 01 (0.97) 240 — — 19 ± 01 (1.21) 26 ± 01 (0.97) 300 — — 16 ± 03 (1.39) 24 ± 01 (1.08) \n Fig. 6 shows the composition of the growing copolyester chains as a function of the reaction time and temperature. At low reaction times, the values of the ratio of PEF to PET unit were much higher confirming the observation that the FDCA reacted very rapidly to produce ‘FDCA-rich oligomers’ followed by incorporation of the TPA into the growing chain. As time progressed, TPA units were incorporated in the growing chains, and eventually expected mole% values were achieved in the case of 250 °C. At 225 °C, the mole% of furan units was higher than the expected values for the time scale studied. This indicated that the incorporation of TPA units was not complete after 300 minutes of esterification reaction time as observed from the Fig. 3(b) . Fig. 6 Mole% of furan in PETF copolyesters determined by 1 H NMR for PETF10 (-) and PETF20 ( ) for direct esterification performed at (a) 250 °C and (b) 225 °C. Dashed lines indicate the expected mole% values (10% for PETF10 and 20% for PETF20). The values of degree of randomness ( R ) at very low times were lower than 1 in some cases indicating a block character for the copolymers. This could be due to the blocks of rapidly esterified FDCA-rich oligomers and absence of PET oligomers formed at low times. However, for most of the reaction time, the R values remained between 1.0 to 1.5 indicating a random microstructure. These R values suggested that even though FDCA reacted faster than TPA, the reversible nature of the esterification reaction caused a redistribution of the repeat units at higher times and randomized the FDCA units in the backbone. Based on the hydroxyl and carboxyl end group conversions and the degree of randomness analysis, it was concluded that the FDCA dissolved in EG almost instantaneously and reacted to form bis-hydroxyethyl 2,5-furandicarboxylate (BHEF) and FDCA-rich oligomers which promoted the dissolution-rate-limited-esterification of TPA with EG most likely by acting as a co-solvent and enhancing the solubility of the TPA in the mixture of EG and oligomers. Note that, the observed improvement in the reaction kinetics of TPA was different for 250 °C compared to 225 °C. This difference was primarily attributed to the temperature effect on the solubility of TPA in EG. At 250 °C, the solubility of TPA in EG was sufficient to promote the reaction and the presence of FDCA-rich oligomers enhanced the dissolution of TPA only marginally. On the other hand, at 225 °C, the esterification kinetics of FDCA was slower compared to 250 °C ( Fig. 4 ). This resulted in a delay time to obtain sufficient concentration of the BHEF and FDCA-rich oligomers to promote the esterification of TPA with EG. Another possible explanation for the observed improvement in TPA conversion was that the dissolved FDCA improved reaction kinetics with catalytic effect since the esterification is an acid-catalyzed reaction. 8 As explained in the following sections, detailed solubility studies and p K a measurements were performed to further investigate the observed improvement in the reaction kinetics of esterification of TPA with EG in the presence of FDCA. Effect of FDCA and pre-synthesized PEF oligomers on the solubility of TPA in EG In a previous study, we reported that the solubility of FDCA in EG is an order of magnitude higher than that of TPA at the esterification temperatures. 31 For PET synthesis, the solubility of TPA in EG is often enhanced by adding small quantities of PET oligomers. 7,8 Solubility experiments were carried out to confirm the proposed hypothesis that BHEF and PEF rich oligomers acted as co-solvents and improved TPA solubility in EG. A fixed quantity of TPA was mixed with different quantities of EG in the presence of FDCA or pre-synthesized PEF oligomers. \n Fig. 7 shows the effect of FDCA and PEF oligomers on the solubility of TPA in moles per kg of EG. Please note that for the experiments with FDCA and PEF oligomers, the weight of TPA was kept constant and additional 20 mole% of FDCA or PEF oligomers was added. The solubility curve of TPA shifted to lower temperatures by ∼10 °C in the presence of 20 mole% pre-synthesized PEF oligomers throughout the temperature range studied. As mentioned previously, similar effect of enhanced solubility of TPA in EG has been reported when PET oligomers were mixed with EG. 8 Fig. 7 Solubility of TPA in moles per kg of EG determined using clear point method for TPA alone (filled triangle, ), TPA (black line) reported by Yamada et al. , 12 TPA with 20 mole% pre-synthesized PEF oligomers (empty circle, ), TPA with 20 mole% FDCA (empty diamond, ), and FDCA reproduced from previous work 31 (filled square, ). The trend in the improvement of solubility was different for the samples with the 20 mole% FDCA. The FDCA did not impact the solubility of TPA at lower temperatures (90 to 140 °C). However, the solubility curve of TPA improved significantly in the presence of FDCA at temperatures higher than 140 °C. The previous work on esterification of FDCA with EG revealed that the onset of esterification reaction of FDCA occurred at 140 °C. 31 Hence, this significant deviation in the solubility of TPA in presence of FDCA at temperatures higher than 140 °C was primarily attributed to the in situ esterified PEF oligomers. However, the improvement in solubility of TPA by in situ esterified PEF oligomers was greater than the improvement observed in the case of TPA with pre-synthesized PEF oligomers. MALDI-MS was employed to investigate the difference in the in situ esterified PEF oligomers and the pre-synthesized PEF oligomers. As confirmed from the MALDI-MS spectrum in Fig. S10 in ESI, † the primary difference was the degree of polymerization. In the case of in situ esterification, most of FDCA was only converted to BHEF and PEF dimer due to the huge excess of EG. Hence, it was hypothesized that the presence of the BHEF formed during the heating step had a greater effect on solubility of TPA in EG compared to the pre-synthesized PEF oligomers with higher degree of polymerization. As previously discussed, during the copolymerization reaction with 10 and 20 mole% FDCA, the instantaneously dissolved FDCA reacted with large molar excess of EG (relative to moles of FDCA) most likely will form BHEF at shorter reaction times (oligomers are expected to form as the reaction progresses since the molar excess of EG relative to moles of FDCA is reduced due to reaction with TPA) which resulted in the observed improvement in TPA conversions. Solubility parameters for the monomers and oligomers were calculated using the group contribution method devised by Hoftyzer and Van Krevelen 33 to support the hypothesis of difference in solubility of TPA in EG in the presence of BHEF vs. PEF oligomers. For consistency, molar volumes of the monomers and oligomers were predicted using the group contribution method by Fedors. 33 Table S2 from ESI † shows the calculated solubility parameters for different chemical species involved in the esterification reaction. Based on the calculated solubility parameters, potential of mutual solubility of two species can be determined from the individual components using eqn (5) . 5 Δ δ 1,2 = [( δ d,1 − δ d,2 ) 2 + ( δ p,1 − δ p,2 ) 2 + ( δ h,1 − δ h,2 ) 2 ] The lower the value of Δ δ 1,2 , the greater the solubility of two components with each other. Δ δ EG, x and Δ δ TPA, x were calculated to understand the solubility of EG and TPA with the different chemical species. 37 Fig. 8(a) and (b) show the calculated Δ δ values for EG and TPA, respectively. As shown in Fig. 8(a) , lower value of Δ δ for the EG,FDCA pair compared to EG,TPA pair indicates that FDCA is expected to have higher solubility in EG compared to TPA. These results are consistent with the observed solubility values reported in Fig. 7 . Similarly, the solubility of BHEF in EG was predicted to be higher than the PEF pentamer based on the Δ δ values calculated for the respective pairs. Even though the BHEF and PEF pentamer have similar δ d and δ p values, the hydrogen bonding contribution ( δ h ) was higher for BHEF compared to PEF tetramer (Table S2 † ). This difference in the hydrogen bonding contribution should result in improved solubility of the EG,BHEF pair relative to the EG,PEF tetramer pair. Additionally, TPA is predicted to have much higher solubility in the BHEF and PEF pentamer compared to EG ( Fig. 8(b) ). Hence, based on the solubility parameter analysis, it was concluded that the sequential nature of the co-esterification on PETF copolyesters with FDCA reacting prior to TPA resulted in improved solubility of TPA in EG due to the presence of BHEF and PEF oligomers in the reaction mixture. The solubility studies revealed that, for the temperature range considered, copolymerization with FDCA would result in a better solubility enhancement than the direct addition of pre-synthesized PEF oligomers primarily due to better solubility of BHEF in EG over PEF oligomers. Fig. 8 Δ δ values calculated using eqn (1) to predict solubility of different reaction species with (a) EG and (b) TPA. Prediction of p K a of FDCA and TPA Direct esterification of carboxylic acids with glycols is an acid-catalyzed Fischer esterification reaction. 8 The carboxylic acid can auto-catalyze the reaction depending on the strength or the p K a of the acid. The p K a value determines the equilibrium between the acid and its conjugate base with lower values of p K a indicating that the acid will be present in the deprotonated form at neutral pH. These free protons can auto catalyze the esterification reaction. 8 Hence, p K a of the diacid can influence the esterification reaction kinetics. In the case of PET, direct esterification of TPA with EG has been reported to be auto-catalyzed by TPA. 7,8 Otton and Ratton reported that the esterification reaction rate increased linearly with decreasing the p K a of the carboxylic acid. 38 The objective of this section was to predict the p K a for FDCA and compare it with the reported values of p K a for TPA. Different predictive tools are available for calculating the p K a s based on the structure. The accuracy of the predictions has been reported to be in good agreement with the experimental values in the case of carboxylic acids. 39 Chemicalize tool provided by ChemAxon was used for p K a predictions for TPA and FDCA. The predicted p K a values for TPA were very close to the experimental values reported in the literature as shown in Table 2 . The predicted values for FDCA were lower than TPA indicating that FDCA is a stronger diacid compared to TPA. Similar trend was observed in the case of monoesterified TPA and FDCA. Based on these predicted p K a values and extending the observation by Otton and Ratton, 38 FDCA should exhibit higher esterification rates than TPA. In conjunction with the reported esterification kinetics data, these results indicate that the improved solubility and faster esterification kinetics are responsible for instantaneous dissolution and conversion of FDCA for PETF copolyesters. The dissolved esterified products of FDCA increase the solubility and potentially reactivity of the dissolved TPA and result in improved TPA conversions at the reaction temperatures studied. A detailed modeling of the dissolution and reaction kinetics should be used to fit the experimental data and obtain the esterification rates to confirm the effect of p K a of FDCA on the esterification rates. However, due to the complexity of the copolymerization process, modeling of the reactions was considered outside of the scope of the current study. Predicted values of p K a for terephthalic acid (TPA) and 2,5-furandicarboxylic acid (FDCA) and comparison to reported values Species Structure p K a Source TPA \n \n \n a : 3.51 \n 38 \n \n b : 4.82 \n a : 3.32 ChemAxon \n b : 4.56 Monoesterified TPA \n \n \n a : 3.61 ChemAxon \n b : 15.10 FDCA \n \n \n a : 2.76 ChemAxon \n b : 3.47 Monoesterified FDCA \n \n \n a : 3.06 ChemAxon \n b : 15.10 End group conversion by titration To confirm the NMR methods used above, the carboxyl end group conversions were determined by titration and compared with the values obtained by NMR as shown in Table 3 . The percent difference between the methods calculated using the eqn (6) was ±13% validating the end group analysis studies performed based on the NMR method. 6 Number average molecular weight ( M n ), dispersity ( Đ ) and X COOH,total calculated with NMR and titration and % difference between the methods as a function of reaction temperature ( T ) and time ( t ) \n T (°C) \n t (min) Sample \n M \n n (Da) \n Đ \n \n X \n COOH,total by NMR \n X \n COOH,total by titration % difference in X COOH,total 250 30 PET 900 1.08 0.57 ± 0.01 0.51 ± 0.01 11.1 PETF10 960 1.07 0.67 ± 0.01 0.64 ± 0.01 5.2 PETF20 960 1.08 0.68 ± 0.01 0.59 ± 0.01 12.6 60 PET 890 1.08 0.84 ± 0.01 0.84 ± 0.04 0 PETF10 970 1.08 0.86 ± 0.01 0.80 ± 0.03 7.3 PETF20 960 1.09 0.89 ± 0.01 0.80 ± 0.03 10.4 225 180 PET 870 1.07 0.55 ± 0.01 0.49 ± 0.03 11.5 PETF10 880 1.09 0.59 ± 0.01 0.66 ± 0.01 −11.1 PETF20 970 1.07 0.75 ± 0.02 0.68 ± 0.01 9.9 420 PET 920 1.08 0.76 ± 0.02 0.81 ± 0.04 −6.7 PETF10 950 1.09 0.85 ± 0.02 0.81 ± 0.01 4.6 PETF20 970 1.08 0.92 ± 0.01 0.86 ± 0.02 6.7 The trend of increase in the end group conversion for PETF samples was consistent in the end group data by titration. The observed variability between the two methods was consistent with the literature reports 40 and was primarily attributed to the random error arising from the inherent differences in the techniques. Molecular weight evolution by MALDI-MS MALDI-MS spectra were recorded to monitor the effect of copolymerization on the number average molecular weight ( M n ) and dispersity or polydispersity index of the samples ( Đ ). Two different reaction times corresponding to X COOH,total for PET of approximately 0.5 and 0.8 were chosen for each reaction temperature as shown in Table 3 . The recorded MALDI-MS spectra are shown in the ESI. † At both reaction temperatures, copolymerization with FDCA marginally increased the number average molecular weight of the oligomers after a reaction time corresponding to X COOH,total ∼ 0.5. The M n remained unchanged at higher reaction times. The dispersity ( Đ ) was close to 1.08 and was unchanged by the reaction time, temperature, or presence of FDCA. The low values of Đ indicated the effective redistribution of repeat units in growing chains as expected in the case of low molecular weight oligomers. It has been reported that the esterification step produces oligomers with 4 to 5 repeat units. 7 Any further increase in the molecular weight needs an effective catalyst system and removal of EG which only takes place in the polycondensation step. The degree of polymerization for the samples was between 4 and 5 (repeat unit molecular weight = 192) consistent with the reports. 7 Hence this data confirmed that the presence of FDCA simply improved the kinetics of the esterification reaction without affecting the degree of polymerization of the oligomers. Alkaline hydrolysis of PETF20 vs. PET This work demonstrated that the solubility and p K a differences in FDCA and TPA result in improved esterification kinetics. Considering the reversible nature of condensation reactions, it was hypothesized that a similar improvement in kinetics may be observed in the depolymerization of PETF copolymers via an alkaline hydrolysis pathway. As a proof-of-concept, a simple alkaline hydrolysis experiment was carried out on polymer (PET and PETF20) film flakes in a glass vial as described in the Experimental section. Due to the nature of the glass vial set up, the reactions were carried out at low temperatures (lower than 100 °C to avoid boiling of the water). To achieve equilibrium conversions at lower temperatures, reaction was run for an extended period of time (3 days). After the workup, the solid residue was weighed to determine monomer molar yield and conversion of PET using eqn (7) and (8) . 7 8 The % conversion and monomer yield of PET and PETF20 depolymerization reactions are shown in Fig. 9 . Both, the conversion and yield almost doubled for PETF20 flake samples compared to PET. 1 H NMR spectroscopy was employed to confirm the purity of the diacid monomers recovered after the depolymerization (Fig. S13 in ESI † ). NMR spectrum confirms that depolymerization was complete and both, FDCA and TPA were recovered as a mixture. Fig. 9 % Conversion ( ) and % diacid yield ( ) of PET vs. PETF20 flakes after alkaline hydrolysis with 1.1 M NaOH solution for 3 days at 90 °C. PET depolymerization reactions are known to be surface reactions, where the alkali, in this case, NaOH, reacts with the surface ester linkages and continues to scrape away the surface of the flakes. 41 Based on the well-proven surface mechanism of depolymerization and the solubility work done in this study we propose the following possible explanations for the observed increase in depolymerization kinetics in case of PETF20, (1) higher solubility parameter of PETF20 enhances the interaction of solvent molecules with the polymer flakes, swelling the polymer matrix resulting in faster reaction kinetics; (2) the ester linkage with FDCA may be more labile to hydrolytic degradation resulting in more rapid depolymerization. Similar effect of higher hydrolytic degradation has been reported in the case of other bio-based co-monomers like isosorbide. 42 A more detailed study of alkaline hydrolysis of copolyester of PET and PEF will be presented in a future paper."
} | 8,982 |
34636665 | PMC8510535 | pmc | 4,212 | {
"abstract": "ABSTRACT Soil microorganisms, which intricately link to ecosystem functions, are pivotal for the ecological restoration of heavy metal-contaminated soil. Despite the importance of rare and abundant microbial taxa in maintaining soil ecological function, the taxonomic and functional changes in rare and abundant communities during in situ chemical stabilization of cadmium (Cd)-contaminated soil and their contributions to the restoration of ecosystem functions remain elusive. Here, a 3-year field experiment was conducted to assess the effects of five soil amendments (CaCO 3 as well as biochar and rice straw, individually or in combination with CaCO 3 ) on rare and abundant microbial communities. The rare bacterial community exhibited a narrower niche breadth to soil pH and Cd speciation than the abundant community and was more sensitive to environmental changes altered by different soil amendments. However, soil amendments had comparable impacts on rare and abundant fungal communities. The assemblies of rare and abundant bacterial communities were dominated by variable selection and stochastic processes (dispersal limitation and undominated processes), respectively, while assemblies of both rare and abundant fungal communities were governed by dispersal limitation. Changes in soil pH, Cd speciation, and soil organic matter (SOM) by soil amendments may play essential roles in community assembly of rare bacterial taxa. Furthermore, the restored ecosystem multifunctionality by different amendments was closely related to the recovery of specific keystone species, especially rare bacterial taxa ( Gemmatimonadaceae and Haliangiaceae ) and rare fungal taxa ( Ascomycota ). Together, our results highlight the distinct responses of rare and abundant microbial taxa to soil amendments and their linkage with ecosystem multifunctionality. IMPORTANCE Understanding the ecological roles of rare and abundant species in the restoration of soil ecosystem functions is crucial to remediation of heavy metal-polluted soil. Our study assessed the efficiencies of five commonly used soil amendments on recovery of ecosystem multifunctionality and emphasized the relative contributions of rare and abundant microbial communities to ecosystem multifunctionality. We found great discrepancies in community composition, assembly, niche breadth, and environmental responses between rare and abundant communities during in situ chemical stabilization of Cd-contaminated soil. Application of different soil amendments triggered recovery of specific key microbial species, which were highly related to ecosystem multifunctionality. Together, our results highlighted the importance of rare bacterial as well as rare and abundant fungal communities underpinning restoration of soil ecosystem multifunctionality during the Cd stabilization process.",
"introduction": "INTRODUCTION Intense anthropogenic activities and rapid industrialization accelerate heavy metal pollution in agricultural soil, leading to a great threat to global food security, ecosystem, and human health. Cadmium (Cd), in particular, a nonessential toxic metal that ranks 7th among 20 strong toxins, is one of the most concerned priority pollutants due to its high risk of human exposure and long residence time in soil ( 1 ). At present, the widespread occurrence of Cd contamination in agricultural soils has been reported in many regions of the world, including Thailand, India, China, and Japan ( 2 ). In China, approximately 1.3 × 10 5 ha of farmlands is contaminated by Cd, accounting for 20% of the total farmland area ( 3 ). With increasing calls for restoration of Cd-contaminated agricultural soil, research efforts have been made to find sustainable and effective remedial solutions over the past few decades ( 2 – 4 ). Compared to physical and biological remediation strategies (e.g., soil mixing, electrokinetic, phytoremediation, and microbial remediation), in situ chemical stabilization has been widely used in the remediation of Cd-contaminated soils due to its efficiency and low-cost in decreasing Cd toxicity and bioavailability ( 5 ). The choice and application strategies of Cd-stabilizing agents are of particular importance for Cd stabilization efficiency in situ since their properties and underlying stabilizing mechanisms vary greatly. Organic amendments (such as biochar, compost, and straw) stabilize Cd and other metals in soil via forming stable organic ligand-metal complexes ( 4 ). Liming materials (such as limestone and calcium hydroxide) can effectively stabilize most metals in soil by increasing soil pH and negatively charged sorption sites of soil colloid and organic matter ( 6 ). The application of clay materials (such as sepiolite and zeolite) to Cd stabilization is mainly based on their high surface areas and excellent ion exchange capacities ( 7 ). Among various Cd stabilizing agents, limestone (primarily CaCO 3 ), biochar, and crop straw are highly recommended in previous studies due to their multiple effects on soil restoration, including reducing Cd bioavailability, alleviation of soil acidification, and enhancing soil ecological functions ( 8 , 9 ). To achieve a better performance, combinations of different amendments are also recommended ( 10 ). Diverse microorganisms in soil play critical roles in maintaining multiple ecosystem functions simultaneously (“ecosystem multifunctionality” hereafter), including nutrients cycling, organic matter decomposition, soil health, and crop productivity ( 11 ). In natural environments, the abundance and distribution of species in microbial communities is uneven, with a few abundant species and a large number of rare species ( 12 ). Traditional studies mainly focus on the abundant members of microbial communities due to their contributions to biomass and nutrient cycling in ecosystems ( 13 , 14 ). However, recent studies have emphasized the ecological importance of rare taxa in maintaining microbial diversity and ecosystem function ( 15 , 16 ). As part of the microbial “seed bank,” rare species exhibit high diversity and functional redundancy and, thus, serve as functional insurance in microbial community ( 17 ). Both abundant and rare species interact intensively, either intra or interkingdom and constitute complex ecological networks. Some species, regardless of their abundance, occupy key positions (e.g., hubs and connectors) in the ecological networks and are considered as keystone species essential for the stability of community structure ( 18 ). Recently, network analysis-based approaches have been used to infer the potential interactions, identify keystone taxa, and decipher the relationship between ecological clustering and environmental factors in many ecosystems ( 19 – 21 ). The keystone species have been shown to be closely pertinent to attributes or functional genes involved in multiple ecological processes, including nutrient cycling, carbon turnover, and crop productivity ( 19 , 22 ). In particular, the rare taxa may function as keystone species responsible for the maintenance of community structure and ecosystem multifunctionality ( 23 ). The responses of abundant and rare species to environmental disturbances are not always consistent ( 24 , 25 ). Abundant species normally occupy a wider niche breadth and can utilize more types of resources, which enable them to be more adaptive to environmental changes than rare species ( 26 ). For instance, due to the discrepancy in resistance to heavy metals, nearly all rare taxa in pristine soil were eliminated by heavy metal pollution, leading to a severe reduction of bacterial diversity ( 27 ). However, contradictory results were also reported in other studies showing that the diversity and community composition of rare taxa are more stable when suffering climate change ( 25 ) and other disturbances, such as copper stress, heat shock, freezing-thawing, and mechanical disturbance ( 28 ). These unaffected rare taxa might be dormant or extremely slow growing but could be activated or become dominant when the environment is favorable ( 28 , 29 ). In addition, distinct assembly processes of abundant and rare communities have been found in many ecosystems, likely due to their differential responses to environmental changes ( 30 , 31 ). During in situ chemical stabilization process, applications of stabilizing amendments lead to multiple changes in soil properties, including metal speciation, soil pH, and available nutrients ( 32 ). These changes may consequently alter the assembly and distribution patterns of abundant and rare species in the microbial community, leading to unknown outcomes for ecosystem multifunctionality. Given that the abundant and rare species may differentially affect functional attributes, distinguishing the roles of abundant and rare taxa in restoration of ecosystem multifunctionality in Cd-contaminated soil is of importance but remains largely unexplored. We hypothesize that the rare community could be more sensitive to amendment-induced changes in Cd bioavailability and soil properties than the abundant community, and the recovery of rare taxa may play vital roles in restoration of soil ecosystem multifunctionality. To test our hypothesis, we conducted a 3-year field experiment applied with five soil amendments (CaCO 3 as well as biochar and rice straw, individually or in combination with CaCO 3 ). The impacts of amendments on composition shifts, niche breadth, and assembly processes of microbial abundant and rare communities were characterized to uncover microbial responses and the mechanisms underlying amendment-induced effects on ecosystem multifunctionality. In particular, we aimed to (i) compare the responses of abundant and rare taxa of bacterial and fungal communities to different soil amendments, (ii) evaluate their contributions to soil ecosystem multifunctionality, and (iii) identify keystone species of abundant and rare communities, which are associated with soil ecosystem multifunctionality in different stabilizing treatments.",
"discussion": "DISCUSSION Distinct responses of microbial rare and abundant communities to soil amendments. Understanding the taxonomic and functional changes of rare and abundant communities in response to soil amendments is of great importance for disentangling microbial processes during in situ chemical stabilization. Consistent with previous studies ( 24 , 33 ), the α-diversity of both rare bacterial and fungal communities was obviously higher than abundant communities (see Table S1 in the supplemental material). However, the application of various amendments did not affect α-diversity but markedly altered the community structure of both rare and abundant taxa ( Fig. 3 ). The greater variations in community similarity of rare bacterial taxa between different treatments confirmed our hypothesis that the rare bacterial community was more sensitive to soil amendments than the abundant community ( Fig. 3 ). This result is in line with previous studies showing greater variations in β-diversity of rare bacterial community than those of the abundant community under environmental disturbances ( 34 , 35 ). The sensitivity of the rare bacterial community could be explained by their narrow environmental breadths to environmental changes ( 36 ). In this study, soil pH and Cd speciation exerted greater impacts on the rare bacterial community than the abundant community (see Table S2 in the supplemental material). Further, the environmental breadths of the rare bacterial community were narrower to soil pH and Cd speciation (see Fig. S2 in the supplemental material). The critical roles of soil pH in regulating microbial community have been emphasized in many previous studies ( 37 , 38 ). During the stabilization process, changes in soil pH are highly related to Cd availability in soil, and the latter has also been reported to affect soil microorganisms ( 39 ). However, our observations demonstrated that regulations of soil pH and Cd speciation on rare bacterial taxa were stronger than the abundant taxa. In contrast to bacteria, the impacts of soil amendments on rare and abundant fungal communities were comparable. Triple application of CaCO 3 together with biochar yielded the greatest variations in rare and abundant fungal communities, which could be also due to the changes in soil pH and Cd speciation. This explanation was supported by a Monte Carlo permutation test between edaphic factors and fungal communities, showing that abundant fungal community was more affected by soil pH and Cd speciation ( Table 1 ). Importantly, a broader environmental breadth of rare fungal taxa to labile Cd fractions suggested that rare fungal taxa were more resistant to Cd stress and could act as a seed bank to sustain ecological functions in Cd-contaminated soils ( 15 ). A similar result has been reported in a previous work documenting that rare fungal taxa are more stable than abundant taxa under different fertilization practices ( 40 ). In addition to soil pH and Cd speciation, the abundant fungal community was also more sensitive to DOC ( Table 1 ; Fig. S2 ). It is reasonable since many fungi prefer soil rich in nutrients and organic matter ( 41 ). Environmental filtering structured the assembly of rare bacterial community. Quantifying the relative contributions of deterministic and stochastic processes to microbial community assembly is a key issue to understand forces structuring community composition ( 42 ). In this study, we found that deterministic assembly was dominant in the rare bacterial community, while stochastic processes primarily governed the abundant bacterial community ( Fig. 4C ). Similar observations have been documented in agricultural fields ( 43 ) and coastal wetlands ( 31 ). The distinct assembly processes between rare and abundant bacterial communities could be due to discrepancies in response and niche breadth to environmental disturbances. It is possible that the rare and abundant taxa occupy distinct ecological niches, which determine their different responses to environmental disturbances ( 35 ). Rare bacterial taxa are more likely to be eliminated by environmental filtering due to their narrow niche breadth, while the abundant taxa occupying a broad niche breadth are more resistant to environmental changes ( 44 ). Therefore, a narrower niche breadth of rare bacterial community to soil pH and Cd speciation may explain our observation that variable selections govern the assembly of the rare bacterial community ( Table S2 ; Fig. S2 ). Despite increasing knowledge on the importance of soil pH and organic matter in bacterial community assembly processes ( 38 , 45 ), our study highlighted that the assembly of the rare bacterial community is more affected by soil pH and Cd speciation, while SOM is crucial for abundant and rare bacterial community assembly processes. Moreover, the significantly lower SES.MNTD values of the rare community indicated a closer phylogenetic clustering by environmental filtering than the abundant community (see Fig. S1A in the supplemental material; P < 0.05). Taken together, application of soil amendments altered soil pH, Cd speciation, and organic matter and could further influence community assembly of rare and abundant bacterial taxa. In line with previous studies showing that the fungal community demonstrates a stronger dispersal limitation than the bacterial community ( 46 , 47 ), here, we found that the assembly of both abundant and rare fungal communities was dominated by dispersal limitation ( Fig. 4D ). This is because fungi are more likely to be limited in long‐distance dispersal compared to the smaller‐sized bacteria, as body size of organisms influences their dispersal ability and spatial aggregation ( 46 ). However, our result was in contrast to a previous study showing that assembly of rare fungal community was dominated by deterministic process in the agricultural ecosystem ( 43 ), possibly due to the differences in habitats and geography. Relative importance of rare and abundant microbial taxa in ecosystem multifunctionality. Restoration of soil ecological function is of importance when assessing the efficiency of in situ stabilization strategies ( 48 ). In the present study, the evidence from the field trial revealed that repeated application of soil amendments (such as CaCO 3 and mixture of CaCO 3 with biochar/straw) promoted the recovery of soil ecosystem multifunctionality ( Fig. 1A ). Spearman correlation showed that ecosystem multifunctionality had a positive correlation with soil pH and strongly negative correlations with labile Cd fractions. These findings suggested that alleviation of soil acidification and Cd toxicity by soil amendments might contribute to the enhanced ecosystem multifunctionality ( Fig. 1B ). Considering distinct responses of microbial rare and abundant communities to soil pH and Cd toxicity, we further investigated their relative contributions to ecosystem multifunctionality. Compared to the abundant bacterial community, the rare bacterial community showed a stronger correlation with ecosystem multifunctionality ( Fig. 1C ). Likewise, a high proportion of rare bacterial keystone species in network analysis further implied the importance of rare taxa ( Fig. 5 ; see also Table S3 in the supplemental material). Meanwhile, we found that both rare and abundant fungal communities were crucial to maintain ecosystem multifunctionality. It is reasonable because fungal species are normally more resistant to heavy metal pollution and play important roles in regulating the ecological functions of contaminated soils ( 49 ). In contrast to previous studies showing that ecosystem multifunctionality is highly related to soil microbial diversity ( 17 , 50 ), we found that the enhanced ecosystem multifunctionality by soil amendments was not assigned to changes in microbial diversity (Spearman correlation, P < 0.05) but due to successions of certain key microbial species. As shown in the distribution of keystone species in different treatments, applications of soil amendments triggered recovery of specific keystone species ( Fig. 5B ). For instance, triple application of CaCO 3 induced enrichment of three rare keystone OTUs, including otu2303 and otu174626 belonging to Gemmatimonadaceae and otu168256 belonging to Haliangiaceae , which were positively correlated with ecosystem multifunctionality. Enrichment of members of Gemmatimonadaceae in soil amended with limestone (primarily CaCO 3 ) has been reported ( 51 ), which are vital species contributing nitrogen cycling and soil respiration in the soil ecosystem ( 52 ). Further, the abundances of these three keystone OTUs showed significantly positive correlations with soil pH but a negative correlation with labile Cd fractions. Together, these results suggest that triple application of CaCO 3 altered soil pH and labile Cd and thereby triggered enrichment of keystone OTUs, which were related to ecosystem multifunctionality. In contrast to CaCO 3 treatment, application of straw decreased Cd availability via ligand exchange of organic matter rather than changing soil pH. Consequently, an enrichment of otu5165 ( Ascomycota ) was observed in triple application of straw treatment. Members in Ascomycota are well known for their ability to degrade lignin and plant residues ( 53 ). In conclusion, this study demonstrated the distinct responses of rare and abundant microbial communities to soil amendments and their relative contributions to ecosystem multifunctionality. Rare bacterial community exhibited greater sensitivity to soil amendments than the abundant community, while the impacts of soil amendments on rare and abundant fungal communities were similar. Soil amendments induced changes in soil pH and Cd speciation and, thereby, influenced the assembly of the rare bacterial community but had limited impacts on the assembly of the abundant bacterial and fungal communities. Furthermore, recovery of specific keystone species by soil amendments may play crucial roles in the restoration of ecosystem multifunctionality in Cd-contaminated soil."
} | 5,054 |
22722235 | null | s2 | 4,213 | {
"abstract": "Microbial ecosystems play an important role in nature. Engineering these systems for industrial, medical, or biotechnological purposes are important pursuits for synthetic biologists and biological engineers moving forward. Here we provide a review of recent progress in engineering natural and synthetic microbial ecosystems. We highlight important forward engineering design principles, theoretical and quantitative models, new experimental and manipulation tools, and possible applications of microbial ecosystem engineering. We argue that simply engineering individual microbes will lead to fragile homogenous populations that are difficult to sustain, especially in highly heterogeneous and unpredictable environments. Instead, engineered microbial ecosystems are likely to be more robust and able to achieve complex tasks at the spatial and temporal resolution needed for truly programmable biology."
} | 226 |
30556001 | PMC6289545 | pmc | 4,214 | {
"abstract": "Single-chamber microbial fuel cells\n(MFCs) were constructed using\nrice bran (carbon source) and pond bottom mud (microbial source).\nThe total electric charge obtained in the MFC combining rice bran\nwith pond bottom mud was four times higher than that in MFC using\nonly rice bran. Phylogenetic analyses revealed dominant growth of\nfermentative bacteria such as Bacteroides and Clostridium species, and exoelectrogenic Geobacter species in the anode biofilms, suggesting\nthat mutualism of these bacteria is a key factor for effective electricity\ngeneration in the MFC. Furthermore, rice bran, consisting of persistent\npolysaccharide, was pretreated by the hydrodynamic cavitation system\nto improve the digestibility and enhance the efficiency in MFC, resulting\nin 26% increase in the total production of electricity.",
"conclusion": "3 Conclusions We constructed\nthe MFC system using rice bran and pond bottom mud.\nElectricity generation from rice bran was significantly improved by\nemploying pond bottom mud as the microbial source. Cooperative action\nof Clostridium , Bacteroides , and Geobacter species would be quite\nimportant in a sequential biological process that includes degradation\nof biopolymers, conversion of saccharides to organic acids, and electricity\ngeneration on anode. Furthermore, the pretreatment of rice bran by\nhydrodynamic cavitation apparently increased the efficiency of MFC.",
"introduction": "1 Introduction Microbial fuel cells (MFCs)\nare promising for energy recovery and\nelectricity generation from organic compounds using microbes as electrocatalysts. 1 , 2 Because of the large variation in microbial metabolisms, many different\norganic compounds can be used as a substrate for electricity generation\nin MFCs. 3 − 5 Many studies have applied MFCs to organic compounds\nderived from food wastes, such as a brewery wastewater, 6 cheese whey, 7 yogurt\nwaste, 8 and palm oil mill effluent. 9 Food waste is useful for electricity generation\nin MFCs because it is rich in organic compounds that can be assimilated\nby microbes. However, most of the above studies used liquid food waste\nand few studies have focused on the solid food waste as a substrate\nfor MFCs. Solid organic compounds are promising substrates for electricity\ngeneration in MFCs because they have higher energy density than soluble\norganic compounds. 3 Rice bran is\na major by-product of rice milling. 10 About\n600 million tons of rice is harvested worldwide annually,\nand rice bran accounts for 7% of the mass of rice. 11 Rice bran has been used for oil production, animal feed,\nfertilizer, and in industrial applications. 12 Recently, rice bran has attracted much attention as a functional\nfood because it contains bioactive ingredients, such as polysaccharides,\nproteins, minerals, and other micronutrients. 10 Rice bran is a potential source material for MFCs because of its\nrich nutrient and organic compounds. 13 However, one problem in the use of solid biomass for MFCs is low\ndigestibility. Rice bran contains cellulose, which has a crystalline\nstructure and is difficult to degrade. Thus, pretreatment of solid\nbiomass is necessary for efficient degradation by microbes in a MFC.\nUltrasonication causes cavitation in a solution, triggering a hotspot\ngeneration with localized extreme temperature and pressure. 14 Ultrasonication has been used to improve the\nperformance of sludge-based MFC for the purpose of pretreatment of\nthe sludge 15 , 16 and removal of the biofilm 17 by the action of the localized high energy.\nA flowing fluid system also gives cavitation, which is called hydrodynamic\ncavitation. Cavitation is generated in a flow of fluid passing through\na venturi tube or an orifice plate with a constriction, resulting\nin hotspot generation. 18 Application of\nhydrodynamic cavitation has been investigated in various fields including\nfood industry and water treatment. 19 We\nhave recently developed a biomass pretreatment technique using hydrodynamic\ncavitation with low energy input and demonstrated a higher pretreatment\nefficiency compared to ultrasonication. 20 In the present study, electricity was generated from solid\nrice\nbran using mud from the bottom of a pond as a bacterial source. A\nsingle chamber MFC equipped with an air cathode was constructed. For\nelectric generation without stirring, an anode was attached to the\nbottom of the MFC reactor, where solid compounds decomposed and electrons\ncollected. The use of hydrodynamic cavitation pretreatment of solid\nrice bran for electricity generation was examined.",
"discussion": "2 Results and Discussion 2.1 Electricity Generation\nUsing Rice Bran in\nMFCs Electricity generation test was conducted using an MFC\nsystem shown in Figure 1 . The voltage was measured for each MFC (MFC-1, MFC-2, and MFC-3)\nduring operation ( Figure 2 ). Initially, the voltage was low in all cases. The voltage\nin MFC-1 (rice bran + bottom mud) increased gradually after day 7.\nRice bran contains macromolecular biopolymers such as cellulose, which\nare gradually degraded to low-molecular-weight compounds by microbes\naround the anode to be assimilated, resulting in a gradual increase\nin the voltage. The higher voltage (325 mV) was achieved on day 15,\nand then it began to decrease. After additional rice bran was supplied\nto MFC-1 on day 20, two peaks showing sharp rise and drastic decline\nin the voltage were observed. The first peak would be attributed to\nthe voltage increase by the assimilation of low-molecular weight compounds\nsuch as sugars and amino acids in additionally supplied biomass. The\nsecond peak could be because of the electricity generation from degradation\nproducts of oligomers such as oligosaccharides and/or peptides. Subsequently,\nthe increase in the voltage was observed again after 30 days, probably\ndue to the degradation of macromolecular compounds such as cellulose\nand proteins. In contrast, the voltage for MFC-2 (rice bran) was quite\nlow. In this system, some degradation and redox reactions catalyzed\nby microbes would occur moderately on the anode and the cathode even\nthough the main microbial source (bottom mud) was not added in MFC-2.\nThe voltage for MFC-3 (bottom mud) was negligible (average voltage\n2.45 mV) throughout the experiment, suggesting that microbes could\nnot grow and generate electricity in this MFC because of the lack\nof organic compounds in the electrolyte. The lowest internal resistance\nand total electric charge are shown in Table 1 . MFC-1 had lower internal resistance (298.7\nΩ) than MFC-2 (1786 Ω) and MFC-3 (9710 Ω). Furthermore,\nthe total electric charge with MFC-1 (1.58 × 10 3 C)\nwas more than four times that of MFC-2 (3.69 × 10 2 C), indicating that MFC-1 performed the best among the three MFCs.\nOn the basis of these results, both rice bran and bottom mud are important\nin our MFC system for electricity generation, where they provide organic\ncompounds and effective biocatalysis, respectively. In terms of power\ndensity, we obtained maximum power density of 16.5 mW/m 2 in MFC-1. Previous works reported power density of 4.2, 21 10, 22 67, 23 37, 24 436 mW/m 2 3 using cattle waste, and 360 mW/m 2 using rice bran, 13 where paddy\nfield soil was used as a microbial source. The actual value of power\ndensity would be significantly and susceptibly affected by several\nfactors such as the performance of anode and cathode including the\nefficiency of metal catalyst, types of substrate (i.e. biomass or\norganic compounds), and microbial source and community employed in\nthe system. As for the types of substrate, electricity generation\nwould be improved by the treatment of biomass as shown in the latter\npart. Figure 1 Schematic diagrams of (A) the air cathode and (B) single chamber\nMFC. Figure 2 Time course of voltage changes in MFC-1, MFC-2,\nand MFC-3. Additional\nrice bran was supplemented to each MFC at the point indicated by the\narrow in the same color. Table 1 Internal Resistance and Total Electric\nCharges MFC-1, MFC-2, and MFC-3 lowest internal resistance r (Ω) total charge\namount (∼day 49) Q (C) MFC-1 298.7 1.58 × 10 3 MFC-2 1876 3.69 × 10 2 MFC-3 9710 2.04 × 10 1 2.2 Phylogenetic Compositions of Bacteria Phylogenetic compositions of bacteria in the original rice bran,\nbottom mud, and anode biofilms of MFC-1 were determined ( Figure 3 ). Chloroplasts made\nup 88% of the rice bran, and were detected using the amplified 16S\nrRNA gene. Thus, bacteria in the class Alphaproteobacteria (10.8%) were predominant in the original rice bran. In the original\nbottom mud, bacteria in the class Betaproteobacteria (18.0%) were dominant as well as the classes Deltaproteobacteria (16.3%) and Alphaproteobacteria (14.7%).\nIn the anode biofilms, bacteria in the classes Bacteroidia (27.5%) and Clostridia (21.4%) were\nenriched compared with the levels in the original bottom mud. Among\nthe bacteria in the class Bacteroidia , the most predominant genus was Bacteroides (37.1% of the class Bacteroidia ).\nSome bacteria belonging to the genus Bacteroides are anaerobic glycolytic bacteria that can produce organic acids\nsuch as acetate and succinate. 25 The bacteria\nin the Clostridia class were mainly\nin the genus Clostridium (61.7%). Many\nkinds of Clostridium species are known\nto be able to decompose cellulosic materials to oligo- and mono-saccharides\nunder anaerobic conditions, 3 suggesting\nthat they would play an important role in decomposing high-molecular-weight\ncompounds in rice bran to low-molecular-weight compounds in the MFC\nsystems. Followed by bacteria in the classes Bacteroidia and Clostridia , bacteria in the class Deltaproteobacteria (13.2%) were enriched in the\nanode biofilms, as was the case with original bottom mud (16.3%).\nAmong the bacteria in the class Deltaproteobacteria , 88.0% belonged to the genus Geobacter . The genus Geobacter are exoelectrogenic bacteria\nwhich use organic acids as a substrate and can directly transfer electrons\nto electrodes without a mediator. 26 , 27 This suggests\nthat bacteria in the genus Geobacter are crucial for voltage generation in MFCs. Furthermore, a recent\nstudy reported phenol-degrading MFCs with graphite electrodes, where Geobacter sp. is working as a phenol degrader in\nthe anode biofilm. 28 Therefore, Geobacter sp. could possibly degrade polyphenolic\ncompounds in the rice bran to generate electricity in our MFC system.\nOn the basis of these results, we hypothesize the following concerted\nelectric generation system; the genus Clostridium degrades polymeric cellulose in rice bran to glucose, some of which\nis converted to organic acids by the genus Bacteroides and/or are directly converted to organic acids by anaerobic Clostridium such as Clostridium butylicum . Finally, these organic acids are used for electron transfer to\nthe electrode by the genus Geobacter . The degrading, fermentative, and exoelectrogenic bacteria would\ncooperatively and sequentially function for effective electricity\ngeneration in the MFC system using rice bran and bottom mud. Figure 3 Phylogenetic\ncompositions of bacteria in (A) rice bran, (B) bottom\nmud, and (C) anode biofilms of MFC-1. 2.3 Effect of Hydrodynamic Cavitation Pretreatment\nof Rice Bran on the MFC Performance Because rice bran contains\na large quantity of biopolymer such as cellulose, these biopolymers\nshould be altered to be easily digested by microbes in the MFC system.\nTo improve the digestibility of biomass, pretreatment is the most\npowerful tool. We thus examine pretreatment of biomass by hydrodynamic\ncavitation, which was found to be efficient for the pretreatment of\nlignocellulosic biomass. 20 Figure 4 A shows the photos of untreated\nand pretreated rice bran by hydrodynamic cavitation. Swelling of the\nrice bran was observed after the pretreatment compared to the untreated.\nThe powder X-ray diffraction pattern of the untreated sample ( Figure 4 B) showed a characteristic\npeak for crystalline cellulose at around θ = 22°. 29 This peak was significantly decreased after\nhydrodynamic cavitation pretreatment, which indicated that the cellulose\ncrystallinity of the rice bran decreased by the treatment. Hot spots\nwith high temperature and high pressure are generated locally in a\nhydrodynamic cavitation system. 18 This\nwould lead to disruption of hydrogen-bond in cellulose in the biomass,\nresulting in decrease in crystallinity. Figure 4 (A) Photographs and (B)\npowder X-ray diffraction patterns of rice\nbran without pretreatment and pretreated by hydrodynamic cavitation\n(at 30 °C for 1 h). Figure 5 shows\nthe\nvoltage of MFC-4 (untreated) and MFC-5 (pretreated by hydrodynamic\ncavitation). MFC-5 (213 mV on day 10) gave higher voltage than MFC-4\n(158 mV on day 11). The total electric charges obtained with MFC-4\nand MFC-5 were 4.91 × 10 2 and 6.19 × 10 2 C, respectively ( Table 2 ), indicating hydrodynamic cavitation increased the total\nelectric charge by 26%. Pretreatment of rice bran by hydrodynamic\ncavitation would increase the digestibility of the biomass by microbes\naround the anode, which leads to higher efficiency in electric generation\nin MFC-5. Pretreatment of biomass was demonstrated to be also important\nin the MFC system. Figure 5 Time course of voltage changes in MFC-4 (untreated) and\nMFC-5 (pretreated\nby hydrodynamic cavitation). The electrolyte was replaced at the points\nindicated with arrows. Table 2 Internal Resistance and Total Electric\nCharges of MFC-4 and MFC-5 lowest internal resistance r (Ω) total charge\namount (∼day 30) Q (C) MFC-4 442.1 4.91 × 10 2 MFC-5 355.1 6.19 × 10 2"
} | 3,387 |
37234113 | PMC10206592 | pmc | 4,215 | {
"abstract": "The progress of the scaffolded DNA origami technology\nhas enabled\nthe construction of various dynamic nanodevices imitating the shapes\nand motions of mechanical elements. To further expand the achievable\nconfigurational changes, the incorporation of multiple movable joints\ninto a single DNA origami structure and their precise control are\ndesired. Here, we propose a multi-reconfigurable 3 × 3 lattice\nstructure consisting of nine frames with rigid four-helix struts connected\nwith flexible 10-nucleotide joints. The configuration of each frame\nis determined by the arbitrarily selected orthogonal pair of signal\nDNAs, resulting in the transformation of the lattice into various\nshapes. We also demonstrated sequential reconfiguration of the nanolattice\nand its assemblies from one into another via an isothermal strand\ndisplacement reaction at physiological temperatures. Our modular and\nscalable design approach could serve as a versatile platform for a\nvariety of applications that require reversible and continuous shape\ncontrol with nanoscale precision.",
"conclusion": "Conclusions In this study, we demonstrated the programmable\nreconfiguration\nof a DNA origami nanolattice via a TMSD reaction. Generally, the construction\nof multiple structural variations from a single DNA origami design\nrequires the replacement of a large number of staple strands because\neven slight changes in the path of the scaffold strand can cause the\nrevision of almost all staple sequences. 12 , 41 This costly preparation has been challenged by the “module-based”\ndesign approach, which enables the construction of a series of DNA\nnanostructures with different morphologies from a single DNA origami\ndesign merely by replacing individual parts of staple strands. 42 − 47 In our design, each frame can be regarded as a module, the cumulative\nreconfiguration of which determines the overall shape. This modular\ndesign enabled a variety of configurations of the nanolattice by dictating\na target shape with a combination of 18 trigger DNAs, which corresponds\nto approximately 8% of the total staple strands only. Furthermore,\nby replacing the incorporated set of the trigger DNAs with another\nvia an isothermal TMSD reaction, the structural shapes could be repeatedly\nand sequentially reconfigured. Given that the properties and\nfunctions of material are tightly\ndependent on its molecular composition as well as the arrangement\nof its constituent molecules, a platform enabling manipulation of\nthe relative positions and postures of multiple molecules with nanoscale\nprecision can be expected to lead to the development of novel materials\nwhose functions can be switched arbitrarily on demand. Owing to their\nsurface addressability, DNA nanostructures have been utilized as scaffolds\nfor organizing a variety of molecules, such as inorganic nanoparticles, 48 , 49 nucleic acids, 50 , 51 and proteins. 52 − 56 Dynamic nanostructures, such as DNA tweezers, capable\nof open/close motion have also been used to control the distance between\na pair of enzyme and its cofactor. 57 Modularity\nof the frame in our dynamic nanolattice should allow its surface decoration\nwith multiple, possibly even different, molecules and their rearrangement\nalong with the reconfiguration of the lattice. In addition, the reconfiguration\ncould be operated at physiologically relevant temperatures (i.e.,\n37 °C), ensuring protection of biomolecules from thermal denaturation.\nCoupled with its scalability, our design approach paves the way for\nconstructing more complex nanosystems whose properties and functions\ncan be controlled in a programmable manner. 58",
"introduction": "Introduction Owing to the development of methods to\nfold DNA into artificial\nnanostructures and evolution of the chemical synthesis of custom oligonucleotides,\nDNA is now widely used as a programmable nanomaterial. 1 , 2 The early stage of the field of the structural DNA nanotechnology\nfocused on the fabrication of static nanostructures with high precision\nin a robust manner. 3 − 5 In addition to that methodology, a variety of dynamic\nDNA nanodevices have been developed based on the differences in physical\nproperties between single-stranded DNA (ssDNA) and double-stranded\nDNA (dsDNA). 6 , 7 ssDNA is often regarded as a flexible\njoint owing to its short persistence length (∼1.3 nm), whereas\ndsDNA has a persistence length of ∼50 nm 8 − 10 and is further\nbundled into rigid parts of the devices. Among the different types\nof methods implemented in structural DNA nanotechnology, scaffolded\nDNA origami is a powerful technique to obtain a desired two-/three-dimensional\nshape 11 − 14 and thus has been employed to construct reconfigurable nanodevices\nimitating normal-sized mechanical objects. 15 − 17 Successful\nattempts are represented by the development of DNA nanodevices and\nnanorobots exhibiting open-close motion, 15 , 18 − 25 sliding motion, 15 , 26 , 27 or rotary motion. 28 − 33 One of the most popular strategies for constructing dynamic\nDNA\nnanodevices is to design flexible connections such as hinges and joints 15 and install angle-adjustable mechanisms for\nthem. 34 , 35 Implementing multiple such connections can\nenhance the complexity of achievable motions of DNA nanodevices. For\nexample, paper-folding-inspired motions were realized by designing\nsix hinges in a DNA origami nanostructure, 36 while kinematic motions were achieved by linking eight-helix bundles\nwith multiple joints. 37 Propagation of\nconformation change based on mechanical linkage was also exhibited\nby controlling one of the angles in a rhombus-shaped nanostructure. 38 Besides these scaffolded DNA origami-based nanodevices,\nreconfigurable grid-like crystalline structures were successfully\nconstructed using a scaffold-free approach by dictating the branching\norientations of their constituent multi-way junctions—achieved\nvia controlling the length of the inter-edge duplexes. 39 , 40 These pioneering studies opened the way for DNA nanodevices exhibiting\nmore complex and controllable motions, toward which operations of\nthe distinct movable joints in a combinatorial, sequential, and reversible\nmanner are further desired. In this study, we designed multiple\ncontrollable links in a single\nDNA origami to produce a multi-reconfigurable nanolattice that can\nbe transformed into a variety of different shapes in a programmable\nmanner depending on the combination of orthogonal input DNA signals.\nThe modular and scalable link design allowed not only reversible reconfiguration\nbut also sequential reconfiguration from one shape into another via\nan isothermal toehold-mediated strand displacement (TMSD) reaction.",
"discussion": "Results and Discussion Nanolattice Design Our DNA origami nanolattice is folded\nfrom a single-stranded M13mp18 scaffold DNA and is constructed as\na 3 × 3 lattice ( Figure 1 a, see also Figures S1 and S2 ).\nEach frame of the nanolattice comprised four rigid four-helix bundles\n(4HBs), four flexible 10 nt ssDNA joints connecting the rigid struts,\nand four latch DNAs, each of which protruded from each 4HB. Reconfiguration\nof the frame was achieved by dictating the angle between the struts\nwith a set of signal DNAs—termed trigger DNAs. Each trigger\nDNA can bridge a pair of latch DNAs to suppress the flexible motion\nof the joints and hold the bundles at predetermined relative angles\n(θ in Figure 1 b). Three orthogonal pairs of trigger DNAs were designed for the\ntransformation of each frame into a right-angled rhombus (θ\n< 90°), left-angled rhombus (θ > 90°), or square\n(θ = 90°) ( Figure 1 b). Each trigger DNA has a toehold sequence and thus can be\ndisplaced from the frame by adding its fully complementary ssDNA (anti-trigger\nDNA) ( Figure S3 ). Figure 1 Design of a DNA origami\nnanolattice comprising shape-controllable\nnine frames. (a) Schematic representation of the DNA origami nanolattice.\nThe latch DNA incorporated into the bundle is shown in green. (b)\nSchematic representation of the transformation mechanism of a frame\n[dashed box in (a)]. One pair of trigger DNAs is used to fix a θ\n= 90° square. A trigger DNA specifically binds the exposed ends\nof the latch DNA to face each other, acting as a sticking rod for\nthe struts (dark gray strands). The other two pairs of trigger DNAs\nare used to fix a θ < 90° or >90° rhombus via\nspecifically\nbringing the attached struts closer together (blue or red strands).\n(c) Transformation of the nanolattice from one shape into another\nby switching the set of trigger/anti-trigger DNAs. The shape of each of the nine frames was regulated\nby their respective\npairs of trigger DNAs, enabling reconfiguration of the nanolattice\ninto various shapes, the interconversions of which were achieved by\nthe TMSD reaction ( Figure 1 c). Transformation of the Nanolattice into Various Shapes Given that each strut (i.e., the 4HBs) of the frame has three patterns\nof angular selectivity (θ < 90°, = 90°, or >90°)\non the two-dimensional (2D) plane, there was a large number of possible\nconfigurations for the nanolattice. Exhaustive search after imposing\nθ ij = 40, 90, or 140° on the\nframe of i th row and j th column\nresulted in 87 possible configurations. These values were estimated\nbased on the length of one trigger DNA and the distance between one\nlatch DNA and one joint. By excluding mirror-image configurations\nand rotationally symmetric configurations, there remained 17 types\nof configurations as apparently distinguishable on the 2D-plane ( Figure S4 ). We first investigated whether\nthese 17 variations could be achieved by the addition of the corresponding\nset of trigger DNAs ( Figure 2 a). For this objective, the unfixed, flexible nanolattice\nwas prepared by one-pot annealing of M13 scaffold DNA and staple strands\nfollowing the removal of excess ssDNA strands ( Figure S5 ). Aliquots of the purified sample were then allowed\nto react with the respective sets of trigger DNAs to produce the 17\ndifferent shapes (NL1–NL17) ( Figure 2 a, see also Figure S6 ). Atomic force microscopy (AFM) images before and after the addition\nof trigger DNAs showed that the unfixed nanolattice adopted various\nshapes, whereas those after the reaction exhibited one distinct shape\n( Figure 2 b, see also Figures S7 and S8 ). Figure 2 AFM analysis of the reconfiguration\ninto 17 possible shapes using\nrespective sets of trigger DNAs. (a) Schematic diagram of the reconfiguration\nfrom an unfixed shape into various shapes. (b) Cropped AFM images\nand histograms of angler distributions. The positions of the nine\nhistograms correspond to the coordinates of each frame in the nanolattice.\nThe background of the histograms was colored based on the average\nvalue of θ ij according to a blue-to-red\ncolor map. Scale bar: 100 nm. N > 100. See also Figures S7–S9 for details. To quantify whether the nanolattice underwent the\nsignal-set-specific\ntransformation, θ ij for individual\nnanolattices were measured, and their distributions were summarized\nin nine (3 × 3) histograms according to their positions ( Figure S7 ). The orientation of the nanolattice\non a substrate surface was distinguished by decorating one of the\nstruts of a specific frame with streptavidin ( Figures S1 and S7 ). Each frame of the unfixed nanolattice\nhad an average angle of approximately 90°, yet exhibited wide\nangle distribution with a large standard deviation, reflecting its\nflexible and polymorphic nature. Following the reaction with trigger\nDNAs, the angle distribution for each frame became sharper, and the\naverage angle matched with the target shape ( Figure 2 b, see also Figures S8 and S9 ). This was applicable in all 17 cases, demonstrating\nthe selective transformation of our nanolattice into the target shape\nusing the specific set of trigger DNAs. Sequential Reconfiguration of the Nanolattice To test\nthe reconfigurability of the nanolattice, the unfixed nanolattice\nwas allowed to react with the specific set of trigger/anti-trigger\nDNAs required to produce a square (NL1), cross (NL3), zigzag (NL15),\nor rhombus (NL17) shape. By sequentially treating the sample with\nthe same or different set of trigger DNAs and their corresponding\nsets of anti-trigger DNAs in order, the transformation was cycled\nfor two rounds. We tested 4 2 = 16 possible pathways, 12\nof which include reconfiguration from a particular shape into others.\nA sample solution of unfixed nanolattice was first prepared (stage\n1) and then allowed to react with the first set of trigger DNAs (stage\n2), in the same manner as presented in Figure 2 . Subsequently, the corresponding set of\nanti-trigger DNAs against the first set of trigger DNAs was added\nto induce the reverse transformation (stage 3). After the reaction,\nthe second set of trigger DNAs was added to induce the second transformation\n(stage 4), and finally, the second set of anti-trigger DNAs (stage\n5) was added to unfix the nanolattice again. Reactions at stages 2–4\nwere conducted at 37 °C for 6 h. After the reaction at each stage,\nthe nanolattice sample was imaged by AFM in solution to analyze the\nchanges in its apparent shape. Figure 3 summarizes the representative AFM images\nafter each stage and the results of the statistical analysis of θ ij . In all pathways, the nanolattices after\nstages 2 and 4 assumed expected configurations with average values\nof θ ij that matched with the target\nshapes, whereas those after stages 1, 3, and 5 assumed indeterminate\nshapes with wide distributions of θ ij ( Figure 3 , see also Figures S10 and S11 ), suggesting the success\nof repeated transformation into a selected shape and programmable\nreconfiguration from one shape to another. Figure 3 Repeated and sequential\nreconfiguration of the nanolattice by adding\nthe corresponding set of trigger/anti-trigger DNAs in order. Flow\nof the reconfiguration from stages 1 to 5 is summarized together with\nrepresentative AFM images and histograms of angler distributions at\neach stage. Unfixed nanolattice was reconfigured into NL1, NL3, NL15,\nor NL17 (from top to bottom) at stage 2 and further reconfigured into\nNL1, NL3, NL15, and NL17 at stage 4. Scale bar: 100 nm. N > 90 for each stage. See also Figures S10 and S11 for details. Hierarchical Self-Assembly into 7 × 7 Nanolattice and Its\nReconfiguration Our design approach should, in principle,\nbe scalable into larger, higher-order structures. To test this idea,\nwe redesigned the nanolattice structure such that its heterotetramerization\nresults in the formation of a 7 × 7 lattice ( Figure 4 a, see also Figure S12 ). Half-struts at the connection ends of each monomer\nwere designed to make complete struts upon the multimerization, resulting\nin the additional frames. Each monomer was first prepared as a 3 ×\n3 square lattice by annealing scaffold DNA and staple strands with\ntrigger DNAs that were required to fix the angles θ between\n4HBs at 90°. Pairs of monomers (A1 and B1 or A2 and B2) were\nthen mixed at an equimolar ratio to produce 7 × 3 lattices, which\nwere further assembled into a 7 × 7 lattice ( Figure 4 a). Figure 4 Hierarchical construction\nof the tetramer of the DNA origami nanolattice\nand its signal-dependent reconfiguration. (a) Schematic illustration\nof the hierarchical assembly of the 7 × 7 nanolattice. (b) Agarose\ngel electrophoresis analysis of the 7 × 7 nanolattice assembly.\nLane 1: ladder maker, lane 2: scaffold DNA, lane 3: A1 3 × 3\nnanolattice, lane 4: B1 3 × 3 nanolattice, lane 5: A2 3 ×\n3 nanolattice, lane 6: B2 3 × 3 nanolattice, lane 7: A1-B1 7\n× 3 nanolattice, lane 8: A2-B2 7 × 3 nanolattice, and lane\n9: 7 × 7 nanolattice consisting of two types of 7 × 3 nanolattices.\n(c) Schematic illustrations of signal DNA-induced reconfiguration\nof the 7 × 7 nanolattice. Representative AFM images at each stage\nare shown. Scale bar: 100 nm. The formation of the target products in each step\nof the hierarchical\nassembly was addressed by agarose gel electrophoresis (AGE). As shown\nin Figure 4 b, two different\nhetero-dimers (A1-B1 and A2-B2) were successfully prepared, as evidenced\nby the decrease of monomer bands and clear band shifts. Their assembly\ninto the heterotetramer was also evidenced by the disappearance of\nthe A1-B1 and A2-B2 bands and further band shifts after the second\nstep. AFM images of the tetrameric sample revealed the successful\nconstruction of the scaled-up, 7 × 7 lattice ( Figure 4 c, left). The 7 ×\n7 lattice was then subjected to the reconfiguration.\nThe anti-trigger DNAs were first added to remove the pre-incorporated\ntrigger DNAs and make the lattice “unfixed” ( Figure 4 c, middle). Next,\nthe unfixed 7 × 7 lattice was reversed into the 7 × 7 square\nlattice or reconfigured into the 7 × 7 rhombus by adding the\ncorresponding set of trigger DNAs ( Figure 4 c, right). Representative AFM images of the\nsample after each step ( Figure S13 ) revealed\nthe transformation of the 7 × 7 lattice into the desired shape,\nwhich also demonstrates the scalability of our design."
} | 4,229 |
30174681 | PMC6107787 | pmc | 4,216 | {
"abstract": "We face major agricultural challenges that remain a threat for global food security. Soil microbes harbor enormous potentials to provide sustainable and economically favorable solutions that could introduce novel approaches to improve agricultural practices and, hence, crop productivity. In this review we give an overview regarding the current state-of-the-art of microbiome research by discussing new technologies and approaches. We also provide insights into fundamental microbiome research that aim to provide a deeper understanding of the dynamics within microbial communities, as well as their interactions with different plant hosts and the environment. We aim to connect all these approaches with potential applications and reflect how we can use microbial communities in modern agricultural systems to realize a more customized and sustainable use of valuable resources (e.g., soil).",
"conclusion": "Conclusion The generation of microbial communities with customized (beneficial) activities has the potential to serve as a powerful approach to enhance sustainable agricultural production by increasing crop health, through combatting plant diseases and reducing the application of fertilizers. To reach this goal a fundamental understanding regarding the functioning of the plant microbiome through microbe–microbe and plant–microbe interaction is required, as well as a deeper understanding of the soil microbial community structure over time (long-term studies) and its plasticity and response to the environmental changes. Also, since individual microbes are key for the regulation of microbial community structure and stability, more comprehensive studies investigating community dynamics using these individual microbes and their soil microbial communities would assist in advancing the field. This knowledge could help to fully understand the impact that these keystone microbes have on crop yields, disease resistance and global nutrient cycles, but also to reveal strategies for microbiome engineering.",
"introduction": "Introduction Soil is considered as one of the most diverse habitats on Earth containing billions of bacteria and millions of fungi (comprising thousands of taxa), as well as larger organisms such as nematodes, ants, or moles ( Bardgett and van der Putten, 2014 ). Recent advances in high throughput sequencing techniques and the increasing number of microbial culture libraries are contributing to show an expanded version of the tree of life dominated by bacterial diversification ( Hug et al., 2016 ). This enormous diversity is driven by the ability of microbes to perform lateral gene transfer across disparate phylogenetic groups ( McDonald and Currie, 2017 ). Moreover, microbial communities are built on high numbers of individuals for each species ( Robbins et al., 2016 ), that can quickly proliferate and have high mutation rates (in the range of 10 −4 in E. coli ) ( Kibota and Lynch, 1996 ; Boe et al., 2000 ; Denamur and Matic, 2006 ) as compared to higher organisms like humans [10 −8 ] ( Kuroki et al., 2006 ; Xue et al., 2015 ). These characteristics increase the diversification of microbes and microbial communities, where individual microbes of the same species could potentially bear different genetic endowments and thus functional characteristics. Soil microbes play key roles in the cycling of nutrients such as nitrogen or phosphorus as well as providing plant protection against biotic and abiotic stress ( Bender et al., 2016 ; Lladó et al., 2017 ). Intensive agriculture has contributed to increases in crop yields but at the same time it has had detrimental effects on the physical and biological properties of soils ( Pimentel et al., 1995 ; Bouwman et al., 2009 ). In intensively managed agricultural systems, the application of fertilizers can compensate for a loss of soil fertility, while tillage disrupts microbial communities ( Johnson et al., 1997 ). This is particularly relevant in the light of current crop production systems with the degradation of more than one half of the global agricultural land while we face massive challenges associated with the disturbance of nitrogen and phosphorous cycles. This situation is very likely to worsen under the prospect of the climate change ( Rockström et al., 2009 ; Yuan et al., 2018 ). As a consequence the United Nations has suggested the re-introduction of sustainable land management practices to minimize land degradation ( Sanz, 2017 ). These practices include crop diversification, use of local adapted species or intercropping in order to maintain soil fertility, carbon sequestration, and nutrient cycling as well as to control soil erosion ( Sanz, 2017 ). Interestingly, these procedures also enhance general soil disease suppression ( Weller et al., 2002 ; Bonilla et al., 2015 ). In addition, sustaining microbial community diversity, structure and composition can help to support ecosystem functions, e.g., by regulating nutrient cycles. During the last decade, microbiome research has modified our perception on the complexity and structure of microbial communities. However, we are only just starting to understand the organization of such complex communities, the interdependencies among themselves and with the biotic (e.g., plant) and abiotic (e.g., edaphic) environment. The increasing need for alternative experimental approaches, as well as the development of new tools has provided new insights into our understanding of the dynamics that occur within the microbiomes and their interaction with host organisms ( Goodrich et al., 2017 ). In studying the human microbiome, the complexity of microbial interactions and the importance of analyzing them separately for each individual has already resulted in novel therapies. Considering the unique microbiome signature of each host, we could move toward a personalized application of microbiome, where we would be able to handle each case independently and better tailor the microbiome to the host’s needs, thus increasing the efficiency of the treatment and the potential of the host ( Human Microbiome Project Consortium, 2012 ). Such “personalized” microbiome approaches would be particularly facilitated by the genetic uniformity of host genotypes of a given crop plant species in the field. Similar to human microbiome studies, there have been efforts to understand the complexity of soil and plant microbiomes ( Bulgarelli et al., 2012 ; Lundberg et al., 2012 ) and to fuel new innovations in sustainable crop production as part of the next green revolution ( Jez et al., 2016 ). However, to exploit the full potential of microbiomes, we require the development of new analytical strategies to comprehend the array of functional capabilities of microbial communities ( Bashiardes et al., 2018 ). The importance of maintaining a diverse and well-balanced microbiome at the plant–soil interface is vital in crop production. Any microbiome applications, however, have to focus on improving key determinants of crop production such as nutrient availability, soil fertility and soil health ( Syed Ab Rahman et al., 2018 ). In this respect, the key challenge is to transfer lab-generated knowledge to the field. In addition to unraveling the structure of the plant/soil microbiome ( Schlaeppi and Bulgarelli, 2015 ), it especially requires us to connect microbial community dynamics with microbiome functioning ( Sánchez-Cañizares et al., 2017 ). In this review we present the challenges and latest efforts that have been made in order to advance our understanding of the different dimensions of microbiomes (e.g., structure, dynamics) and how it affects plants. We further introduce future approaches to access the full potential of the soil microbiome, including beneficial microbes, in improving crop production."
} | 1,944 |
27208877 | null | s2 | 4,217 | {
"abstract": "Once thought to live independently, bacteria are now known to be highly social organisms. Their behaviors ranges from cooperatively forming complex multispecies communities to fiercely competing for resources. Work over the past fifty years has shown that bacteria communicate through diverse mechanisms, such as exchanging diffusible molecules, exporting molecules in membrane vesicles, and interacting through direct cell-cell contact. These methods allow bacteria to sense and respond to other cells around them and coordinate group behavior. In this review, we share the discoveries and lessons learned in the field of bacterial communication with the aim of providing insights to parasitologists and other researchers working on related questions."
} | 188 |
34149646 | PMC8211778 | pmc | 4,219 | {
"abstract": "Nitrogen is one of the limiting nutrients for coral growth and primary productivity. Therefore, the capacity of different associations between corals and their algal symbionts (Symbiodiniaceae) to efficiently exploit the available nitrogen sources will influence their distribution and abundance. Recent studies have advanced our understanding of nitrogen assimilation in reef-building scleractinian (hard) coral-Symbiodiniaceae symbioses. However, the nutrient metabolism of other coral taxa, such as Alcyoniina (soft corals), remains underexplored. Using stable isotope labeling, we investigated the assimilation of dissolved nitrogen (i.e., ammonium, nitrate, and free amino acids) by multiple species of soft and hard corals sampled in the Gulf of Aqaba in shallow (8–10 m) and mesophotic (40–50 m) reefs. Our results show that dissolved nitrogen assimilation rates per tissue biomass were up to 10-fold higher in hard than in soft coral symbioses for all sources of nitrogen. Although such differences in assimilation rates could be linked to the Symbiodiniaceae density, Symbiodiniaceae species, or the C:N ratio of the host and algal symbiont fractions, none of these parameters were different between the two coral taxa. Instead, the lower assimilation rates in soft coral symbioses might be explained by their different nutritional strategy: whereas soft corals may obtain most of their nitrogen via the capture of planktonic prey by the coral host (heterotrophic feeding), hard corals may rely more on dissolved nitrogen assimilation by their algal symbionts to fulfill their needs. This study highlights different nutritional strategies in soft and hard coral symbioses. A higher reliance on heterotrophy may help soft corals to grow in reefs with higher turbidity, which have a high concentration of particles in suspension in seawater. Further, soft corals may benefit from lower dissolved nitrogen assimilation rates in areas with low water quality.",
"introduction": "Introduction Nitrogen (N) is essential for the life and growth of all organisms on Earth, as it is required for the biosynthesis of key cellular components. However, N is a growth-limiting nutrient in oligotrophic marine ecosystems, such as coral reefs ( de Goeij et al., 2013 ). Corals are the primary reef-building and habitat-forming species in these marine environments and require a steady supply of nutrients for growth and reproduction. As a consequence, they have evolved as meta-organisms or holobionts ( Rohwer et al., 2002 ; Bosch and McFall-Ngai, 2011 ), in which the host is associated with an assemblage of microorganisms. These microbial communities are notably involved in the protection against pathogens and in the efficient uptake and recycling of the few available nutrients ( Rädecker et al., 2015 ). The coral animal itself is mainly capable of assimilating particulate and dissolved organic N [e.g., dissolved free amino acids (DFAA) and urea] ( Grover et al., 2006 , 2008 ). However, microbes involved in all steps of the N cycle have been found in the microbiota of corals. For example, N-fixing microorganisms ( Benavides et al., 2017 ) convert N 2 gas into bioavailable ammonium (NH 4 + ), and numerous microbes, including the common hard and soft coral symbiont Endozoicomonas , can perform dissimilatory nitrate (NO 3 – ) reduction into NH 4 + ( Neave et al., 2014 ). Finally, the algal symbionts belonging to the family Symbiodiniaceae ( LaJeunesse et al., 2018 ) are the main assimilation site of the dissolved inorganic N forms (DIN), such as NH 4 + and NO 3 – ( Muscatine et al., 1979 ; Grover et al., 2002 , 2003 ; Pernice et al., 2012 ). The assimilation of dissolved N (DN) by corals is, however, influenced by both the environmental conditions and the algal symbionts they are associated with. For example, some genera of Symbiodiniaceae take up nutrients more efficiently than others ( Baker A.C. et al., 2013 ; Leal et al., 2015 ; Pernice et al., 2015 ), and high light levels promote their DN assimilation ( Grover et al., 2008 ). In contrast, elevated seawater temperatures reduce DIN availability in surface waters due to enhanced water column stratification, which prevents the upwelling of nutrients recycled in deep waters ( Behrenfeld et al., 2006 ). It also induces coral bleaching and thereby impairs the ability of the remaining symbionts to take up DIN ( Godinot et al., 2011 ; Krueger et al., 2018 ). As N assimilation is positively correlated with primary productivity, the capacity of different coral-dinoflagellate symbioses to efficiently exploit the different N sources available will influence their distribution and abundance in a given environment. Therefore, a thorough understanding of the acquisition and allocation of N within different associations and different environments [e.g., shallow (high light) versus mesophotic (low light) reefs] is essential to improve our prediction of the ecological conditions under which each association is favored. However, the nutritional ecology of coral-dinoflagellate symbioses, particularly regarding the N metabolism, has mostly focused on a few main reef-building scleractinian (hard) coral species ( Muscatine et al., 1989 ; Hoegh-Guldberg and Williamson, 1999 ; Mills et al., 2004 ; Baker D.M. et al., 2013 ; Ezzat et al., 2017 ). On the other hand, other coral taxa such as soft corals (sub-order Alcyoniina) have been largely overlooked ( Schlichter, 1982 ; Burris, 1983 ), despite being the second most common benthic group on many reefs and therefore recognized as key taxa ( Schubert et al., 2017 ). The lack of data on soft corals, along with the use of different data normalization metrics in studies on soft and hard corals, limit our ability to predict how nutrient conditions may favor the growth of one group over the other. High abundances of octocorals (soft corals or gorgonians) have, however, been observed on eutrophicated reefs, where high concentrations of nutrients in the water do not favor the growth of hard corals ( Bell, 1992 ; Gast et al., 1999 ; Baum et al., 2016 ; Vollstedt et al., 2020 ). This dominance of soft corals in disturbed reef ecosystems suggests that they may have a different nutrient metabolism or nutritional relationship with their symbionts compared with hard corals. Also, corals tend to increase their reliance on heterotrophy with depth (mesophotic reefs), although this might be species-dependent ( Muscatine and Kaplan, 1994 ). To gain better insights into the extent to which soft corals rely on their symbionts for their nutrition, we investigated how these symbiotic associations assimilate different DN forms, and how this may be impacted by depth and temperature. We hypothesized (1) that Symbiodiniaceae in symbiosis with soft corals can use all DN forms, and (2) that low irradiance (as measured at mesophotic depths) or elevated temperatures will negatively affect the assimilation rates of DN, as previously observed for hard corals. In addition, we conducted a comparative study with hard corals, allowing a better understanding of the differences in the functional and nutritional ecology of soft and hard corals, with the underlying hypothesis that soft corals rely less on the assimilation of DN than hard corals because they are generally considered as more heterotrophic. A higher reliance on heterotrophy may help soft corals to grow in reefs with high turbidity, and they may benefit from low DN assimilation rates in areas with low water quality.",
"discussion": "Discussion Nitrogen availability is a major factor determining the growth, productivity, and overall fitness of coral holobionts ( Rädecker et al., 2015 ). Within these holobionts, the assimilation of inorganic N dissolved in seawater (NH 4 + and NO 3 – ) is mainly performed by their algal symbionts, as they are the most active site for NH 4 + fixation ( Grover et al., 2002 ; Pernice et al., 2012 ), and are the primary site of fixation for NO 3 – , because animals do not possess NO 3 – reductases ( Miller and Yellowlees, 1989 ). Following assimilation, the algal symbionts transfer N to their coral host in the form of organic N compounds ( Rädecker et al., 2015 ). The assimilatory capacity of the endosymbionts depends, however, on the transition of N from seawater through the host tissue to the symbiont cells. In contrast, dissolved organic N forms, such as amino acids (DFAA), can be directly assimilated by both the coral host and algal symbionts into proteins ( Grover et al., 2008 ). Therefore, both partners play a role in N assimilation, which together determines the capacity of the holobiont to take up nutrients. This study highlights a different capacity of soft and hard corals to assimilate dissolved N forms. The lower assimilation rates per unit biomass measured in soft coral holobionts are likely due to different nutritional strategies and morphological traits, such as a lower reliance on the algal symbionts for N acquisition. In addition, we observed an increased assimilation of N with increasing depth or seawater temperature for most but not all coral-Symbiodiniaceae associations. Differences in N Assimilation Between Soft and Hard Coral-Symbiodiniaceae Symbioses Our study highlights 10-fold lower rates of dissolved N assimilation per unit biomass in soft coral holobionts than in hard coral holobionts, whose uptake rates are comparable to previous studies ( Supplementary Figure 3 ; Grover et al., 2002 ; Pernice et al., 2012 ; Ezzat et al., 2017 ). Uptake rates of dissolved nutrients have previously been linked to the Symbiodiniaceae densities or Symbiodiniaceae species, as some are less efficient than others in nutrient acquisition ( Baker A.C. et al., 2013 ; Ezzat et al., 2017 ). In our study, however, the genus and density of Symbiodiniaceae were similar between the soft and hard corals investigated and could thus not explain the differences observed in N assimilation rates. Percentages of translocation of organic N compounds derived from the assimilation of NH 4 + and NO 3 – by the algal symbionts were also not significantly different between the hard and soft coral groups. In addition, the lower assimilation rates of dissolved N (DFAA) in soft corals suggest that they have lower uptake capacities or, alternatively, lower needs for these N sources than hard corals. However, the similar N contents per unit biomass in soft and hard coral tissue and Symbiodiniaceae do not support different N needs in one of the symbiotic associations, although the tissue turnover can be different between the two coral groups. Several morphological and metabolic characteristics can contribute to the higher N assimilation rates in hard coral compared to soft coral symbioses. At the host level, soft corals are primarily characterized by the absence of a calcium carbonate skeleton and only possess sclerites as calcified structures ( Fabricius and Alderslade, 2001 ). In hard corals, the deposition of a skeleton (calcification) generates protons that have to be neutralized to avoid tissue acidification ( Comeau et al., 2013 ). Crossland and Barnes (1974) and Biscéré et al. (2018) suggested that ammonia may be involved in proton neutralization, through the ornithine cycle and urea production, therefore possibly promoting the uptake of DIN by the symbionts. In addition, soft corals comprise a large coenenchyme mostly constituted of mesoglea, which can represent a significant barrier to transepithelial diffusion of molecules (oxygen in Bradfield and Chapman, 1983 ; bicarbonate in Furla et al., 1998 ). The expansion state, together with the low surface area to body volume ratio, usually found for soft corals exhibiting fleshy and massive morphologies, are also not in favor of exchanges through the epidermal tissue ( Kirschner, 1991 ; Shick, 1991 ; Fabricius and Klumpp, 1995 ). At the symbiont level, Symbiodiniaceae associated with hard corals living in shallow waters generally exhibit higher rates of photosynthesis than those in symbiosis with soft corals (e.g., Fabricius and Klumpp, 1995 ; Pupier et al., 2019b ). As N uptake is proportional to the algal symbiont’s photosynthetic activity (e.g., Grover et al., 2002 ), the higher photosynthesis levels of hard coral symbionts ( Pupier et al., 2019b ) can explain their higher rates of N assimilation. Besides Symbiodiniaceae, other eukaryotic as well as prokaryotic microbes may also be involved in the cycling of N within the coral holobiont. For example, “new” N can be introduced via N 2 fixation activity by symbiotic diazotrophic bacteria, but this has so far not been observed in soft corals ( Pupier et al., 2019a ). Soft corals may also harbor a higher abundance of microbes involved in the dissimilatory nitrate reduction to ammonium (DNRA) process, recycling N and in turn providing DIN to their host ( D’Elia and Wiebe, 1990 ). Higher levels of N recycling via DNRA may limit the need for additional “new” N uptake. N may also be lost via denitrification processes, which may alleviate excess N availability in coral holobionts ( Tilstra et al., 2019 ). The microbial role in N assimilation and recycling, both in soft and hard corals, remains to be further investigated. Although microbes may play a role in the N cycling within the coral holobiont, our observations suggest that the contribution of the microbial symbionts to N assimilation is rather limited in soft corals. Soft corals thus likely rely primarily on heterotrophic feeding on particulate organic matter to meet their N requirements. Soft corals, and octocorals in general, are indeed known to capture high amounts of phyto- and zooplankton as well as other forms of particulate organic matter suspended in the surrounding seawater ( Sebens and Koehl, 1984 ; Fabricius et al., 1995a , b , 1998 ; Fabricius and Dommisse, 2000 ; Migné and Davoult, 2002 ; Piccinetti et al., 2016 ). To test the contribution of autotrophy and heterotrophy to the N demand in soft and hard corals, tracers can be applied, such as Compound Specific Isotope Analysis of the amino acids ( Ferrier-Pagès et al., 2021 ). Effect of Depth and Seawater Temperature on N Assimilation Rates Symbiodiniaceae associated with soft and hard corals showed the same trends with depth, regardless of their host species. They either maintained or increased their chlorophyll content (per unit biomass or per symbiont cell) at mesophotic depth, which is likely a strategy to increase the capture of light energy ( Kahng et al., 2019 ) for acclimation to the lower light levels ( Mass et al., 2007 ; Ziegler et al., 2015 ). Also, their translocation of organic N compounds derived from the assimilation of NO 3 – was enhanced at mesophotic depth in three out of the six coral species. This finding is in contrast with previous observations that light stimulates N uptake and assimilation ( Grover et al., 2002 ). Such a discrepancy may be linked to the normalization used (i.e., the surface area in previous studies and the tissue biomass in this study). Finally, the assimilation of DFAA was higher in the host fraction of the hard coral species and in the Symbiodiniaceae of R. f. fulvum at mesophotic depth. Such higher assimilation of organic N corresponds with a reduced reliance on autotrophy due to reducing light levels with increasing depth in mesophotic corals. Consequently, they may rely more on heterotrophy ( Williams et al., 2018 ) while hard corals may also obtain more N from diazotrophic bacteria ( Bednarz et al., 2018 ). Overall, the enhanced assimilation rates of N at mesophotic depth suggest higher N needs in mesophotic colonies to sustain their metabolism, but this hypothesis remains to be further investigated. Increased temperatures tended to enhance the assimilation of N in both the host and algal symbiont fractions of soft corals, which is in agreement with a previous study performed on the N assimilation rates in S. pistillata at high temperatures (29°C) ( Godinot et al., 2011 ). Such increases in N assimilation can be of a mechanistic nature, for example a thermal optimum reached for the enzymes involved in N assimilation. However, it could also be linked to an increased metabolism at high temperature ( Gillooly et al., 2001 ), although this hypothesis remains to be tested. Ecological Implications Overall, this study corroborates that soft coral-Symbiodiniaceae associations depend on another N source than dissolved nitrogenous compounds to meet their N requirements. This suggests that soft corals rely primarily on heterotrophy. This may be especially true for species bearing polyps with low surface area to volume ratios because they have lower photosynthetic rates, and thus rely less on autotrophy, than species bearing polyps with high surface area to volume ratios ( Baker et al., 2015 ; Rossi et al., 2018 ). A lower reliance on their algal symbionts may help soft corals to grow on reefs with higher turbidity or sedimentation regimes, as these conditions decrease the amount of light received by corals (decreased autotrophy) but present high concentrations of suspended particles in the seawater ( McClanahan and Obura, 1997 ). Further, the lower DN assimilation rates by soft coral-Symbiodiniaceae associations may favor their survival in areas with low water quality. For example, the algal symbionts may shift from a N limited to a phosphorus-starved state under excess N availability ( Wiedenmann et al., 2012 ), resulting in phospholipids in the chloroplast’s thylakoid membranes being substituted by sulfolipids. Since phospholipids are essential to the stability of the membranes under heat-stress conditions ( Tchernov et al., 2004 ), increased N availability may therefore increase the bleaching susceptibility of corals ( Wiedenmann et al., 2012 ). In addition, excess N can reduce photosynthate translocation rates by Symbiodiniaceae ( Ezzat et al., 2015 ), leading to the starvation of the coral host. As assimilation rates of N are relatively low in soft corals, it is however unlikely that excess N would enter the tissues and disrupt the symbiosis. This may be a reason why soft corals are more abundant than hard corals on reefs exposed to high concentrations of dissolved inorganic nutrients ( Baum et al., 2016 ). Further studies investigating the N and phosphorus budget of soft and hard coral holobionts along a eutrophication gradient are needed to investigate how nutrients may explain the ecological niches of these two coral groups."
} | 4,626 |
35160847 | PMC8840348 | pmc | 4,222 | {
"abstract": "In this paper, the ballistic damage mechanism and residual bearing capacity of ceramic/backing plate armor were investigated. First, a series of lightweight armors were prepared, consisting of ceramic and ultra-high molecular weight polyethylene fiber-reinforced resin matrix composite (UHMWPE) plates, and were wrapped in a high-strength fabric. Then, the ceramic/UHMWPE armors were hit by one or two bullets, and finally subjected to compression testing. The results showed that the main failure mode of integral ceramic/UHMWPE armors was ceramic brittle fracture. Many zigzag patterns on the compression curve indicated that the specimens had undergone the stages of crack propagation, ceramic fragment reorganization, plastic deformation of UHMWPE backing plate, interlaminar tearing, and overall fracture. The failure of spliced ceramic/UHMWPE armors was mainly due to the dislocation between ceramic sheets; the smooth compression curves indicated that there was no recombination of ceramic fragments and obvious interlayer debonding during the compression. Under the maximum load, each ceramic/UHMWPE armor with ballistic damage did not suddenly break and fail. The structure and thickness of ceramic plates all had an impact on residual strength: under the same structure, the greater the thickness, the greater the residual strength, but the relationship between them was not linear; under the same thickness, the residual strength of the spliced ceramic/UHMWPE armor was higher. The residual strength was also related to the number of shots: after two bullets hit, its value was only one-third of that after one bullet hit.",
"conclusion": "4. Conclusions Based on the principle of being lightweight, a series of ceramic/UHMWPE armors were prepared by controlling the area density so that it did not exceed 4.4 g/cm 2 . Through the shooting test, the bulletproof performance of the designed ceramic/UHMWPE armors was proved to meet the requirements of the American bulletproof standard in MIL-PRF-46103E. Due to the different ceramic size and configurations, the ceramic/UHMWPE armors showed different properties in ballistic damage mechanism, compression failure behavior, and residual bearing capacity. Ceramic plates with different structures showed different ballistic damage modes. The damage to the integral ceramic plate was mainly ceramic crushing due to the action of the stress wave back and forth, the petal-like ceramic cone was finally formed. The main damage form of spliced ceramic armor was the dislocation of ceramic sheets. This is because the preferential movement of adjacent ceramic sheets consumed part of the impact energy, and the residual energy was not enough to lead to the crushing of the ceramic sheets. The configurations of ceramic plates were different, and the ceramic/UHMWPE armors after ballistic tests showed different compression test phenomenon and compression failure modes. The integral ceramic/UHMWPE armor experienced the stages of ceramic fragment reorganization, plastic deformation of the backing plate, interlayer tear, crack propagation, and fracture caused by compressive load; the zigzag phases on the compression curve proved that the ceramic fragments were reorganized to form a pseudo-integral ceramic plate, and then destroyed rapidly again. The spliced ceramic/UHMWPE armor had no reorganization of the ceramic fragments and obvious interlayer debonding. It always exerted its bearing capacity as a whole, which is proved by its smooth compression test curve with a relatively steep slope similar to that of a single uniform material. Under the maximum load, the all ceramic/UHMWPE armors first deformed to a certain extent, and then broke, indicating that the ceramic/UHMWPE armors after bullet shooting damage could still work safely through deformation: win time for the protection of life and property. Residual strength is an absolutely competent parameter in the structural safety assessment and life prediction. The remaining bearing capacity of the ceramic/UHMWPE armor after ballistic damage was affected by the configuration, thickness, and damage mode of the ceramic plate. After a single attack with the same working condition, the residual strength of the spliced ceramic/UHMWPE armor was greater than that of the integral ceramic/UHMWPE armor provided that their total thickness and the plate thickness of each layer was the same. Under the same working condition, after two bullets hit, the residual strength of the spliced ceramic/UHMWPE armor decreased rapidly, which was only one-third of that after one bullet hit. Similarly, after one bullet hit, the greater the thickness of the ceramic plate, whether it was integral or spliced, the greater the residual strength of the composite armor. However, as shown in the experimental data shown in Table 1 , the relationship between the residual strength and the thickness of the ceramic plate does not increase linearly. When preparing protective armor equipment, the requirements of manufacturing cost, service condition, and protection level can be comprehensively considered to design an appropriate thickness of the ceramic plate.",
"introduction": "1. Introduction Ceramic materials have the characteristics of high hardness, low density, wear resistance, but their ability to withstand tension and bending is small. Therefore, when a ceramic material is used to resist bullet penetration, it needs to be equipped with a tough back plate [ 1 ]. Due to its excellent properties such as high temperature resistance and impact resistance, it has especially attracted attention in the defense of armor-piercing firebomb and projectile fragments [ 2 ]. In general, ceramic/backing plate armors consist of a hard brittle ceramic facing and deformable backing materials. The difference in physical properties of each layer interferes with the penetration of the projectile (jet). The ceramic facing the projectile destroys the projectile tip, slows it down and transfers the load to a large area of the backing; the backing supports the ceramic so that the broken ceramics cannot be scattered and sprayed to cause secondary damage. At the same time, the projectile is brought to rest through the good self plastic deformation of the backing [ 3 , 4 , 5 ]. In the fields of armed helicopters, military armored vehicles, and body protective equipment, people have been looking for the most effective way to achieve the optimal armor configuration and the lowest area density as possible, and trying to use lightweight and low-cost ceramic/backing plate armor to resist high-end threats [ 6 , 7 ]. The research on material characteristics, armor structure, and ballistic damage mechanisms has been carried out continuously. Light composite armor is constantly being designed and prepared, and the relationship between the residual penetration depth and the thickness of the ceramic panel has also been revealed [ 8 , 9 ]. The ceramic/backing plate armor is similar to a laminate structure and contains two or more different materials. Compared with metals, the damage propagation law of laminates is difficult to predict. Under impact load, the deformation of the laminate is the reason why it can absorb energy. The laminates preferentially exhibit plastic deformation owing to the absorbance of impact energy [ 10 , 11 , 12 ]. According to the results of shooting experiments, the damage forms found in some ceramic/backing plate armors include: visible plate burst damage, backing plate concave (convex), large deformation damage, interlayer delamination damage, etc. There have also been invisible plastic deformation, micro cracks, and other damages [ 13 , 14 ]. Residual strength refers to the maximum bearing capacity of a cracked structure, which generally depends on the material properties, initial crack length, and service time. The purpose of residual strength measurement is to predict whether the structure can withstand damage tolerance loads under certain damage conditions without catastrophic failure [ 15 , 16 , 17 ]. Residual strength research belongs to the strength research of damaged and defective components and is an important consideration in safety assessment. Through the test and analysis of the actual bearing capacity of damaged and defective structures, a reference can be provided for inferring the service life of the structure. At present, whether in the military or other areas with protection needs, the requirements for protective armor that can ensure the safety of personnel and equipment are becoming higher and higher. For example, a helicopter hit by bombs during a mission needs to return safely, and an armored vehicle attacked by an explosive must still maintain the ability to survive and fight on the battlefield. The residual strength can be used to evaluate the protective ability of composite armor after a bullet attack, providing an effective research method for equipment protection and personnel survival. In this study, the ceramic/backing plate armor samples were prepared by using ceramics as the front plate and ultra-high molecular weight polyethylene (UHMWPE) fiber-reinforced resin matrix composite as the backing plate, here, named as ceramic/UHMWPE armor. There were two types of ceramic plate: integral or spliced. Then, they were arranged for shooting experiments, and a subsequent compressive test; the relative experimental phenomena were analyzed, and the failure mode and crack propagation law were discussed and compared for the different ceramic/UHMWPE armors Finally, residual strength was introduced to access the safety of the ceramic/UHMWPE armors with impact damage, and the affecting factors also were discussed. The present study will provide a methodology and some reference data for obtaining the optimum design of ceramic/UHMWPE armor with high safety performance.",
"discussion": "3. Results and Discussion 3.1. Experimental Analysis of Shooting Test and Ballistic Damage Mechanism The front and back shapes of the ceramic/UHMWPE armors after the shooting experiment are shown in Figure 1 . There is no large area of ceramic peeling off at the impact points; the layers are well bonded without obvious interlayer debonding phenomenon; the overall shape of the specimen remains unchanged, and the frontal impact point corresponds to the back convex position; the targets were not penetrated by the bullets. The heights of the back convex are shown in Table 1 . The results show that the ballistic performance of the ceramic composite armors meets the requirements of the MIL-PRF-46103E ballistic standard [ 23 ]. The bullet marks on the front surface of the armors in Figure 1 a,b are compared. It is found that there is obvious ceramic debris at the impact point of the integral ceramic plate, and the anti-cracking cloth around the impact point is a little convex. Thus, it can be inferred that the ballistic damage mode of the integral ceramic plates after a bullet impact is mainly ceramic crushing and crack propagation. However, the broken ceramic fragments were separated from the integral plate and stuck together with the anti-cracking cloth, which resulted in some uneven zones and bulges on the surface of the anti-cracking cloth Conversely, there is no obvious ceramic debris at the impact point of the spliced ceramic plate, the anti-cracking cloth around the impact point is flat, and there is only some less obvious indentation on the surface. It is speculated that the damage of the spliced ceramic plate after a bullet impact is mainly due to the dislocation between the small ceramic sheets; the impact force on the impact point spreads to the adjacent ceramic sheets, and the change of their relative position consumes a lot of energy, which reduces the tendency of the ceramic sheets to break. The indentation on the anti-cracking cloth is caused by the change in the relative position between the ceramic sheets. The shape and size of the back convex lumps in Figure 1 a,b are compared. It is found that the area and height of the convex on the back surface of the integral ceramic target are small, while those on the back surface of the spliced ceramic target are relatively large. This can be explained by the damage mechanism of the ceramic plate. The damage of the integral ceramic plate is mainly fragmentation, most of the impact energy is propagated along the radial, circumferential, and thickness directions in the ceramic plate, and a lesser part is transferred to the backing plate, so the back convex area and height are smaller. The spliced ceramic plate absorbs the impact energy mainly through the dislocation between the ceramic sheets when the target is impacted. However, the number of dislocations is limited, and the energy absorbed by the dislocation is also limited. Part of the energy is easily transferred to the backing plate, so there is a big bulge on its back surface. 3.2. Experimental Analysis of Compression Test and Failure Mechanism Considering the diversity of the damage modes of composite armor materials, it is difficult to select simple characterization parameters such as crack length to determine the damage degree. Therefore, the residual strength evaluation test method of composite laminates after impact in ASTM D7137/D7137M-2017 standard was used to conduct compression failure tests on the composite armor specimens with impact damage after target shooting [ 25 ]. Their damage propagation processes, deformation modes, and final failure modes during compression failure were analyzed and compared. Finally, the residual strengths were calculated [ 26 ]. Figure 2 shows the designed anti-instability support fixture and its application in the compression test. The spacing between the components in the support fixture can be adjusted according to the size of the test sample, and a set of vertical guide plates are added to restrain the tested sample, so as to avoid instability or bending of the sample during loading. The compression test was carried out at room temperature. The samples were slowly loaded and the load/displacement curves were observed on the computer screen. During the whole loading process, the fixture was not damaged, such as loosening and tilting, and the ceramic composite target samples also did not undergo deflection, distortion, or other unstable phenomena, indicating that the test process was reliable and the test data were valid. Because the anti-cracking layer was wrapped outside the target plate, the crack propagation in the target plate during the compression failure process could not be seen immediately. The failure process was only judged by carefully listening to the sound of crack propagation. The actual situation of crack propagation could be observed only after the experiment was completed and the anti-cracking layer was removed. 3.2.1. Composite Armors with Integral Ceramic Plate (1) Experimental phenomena and analysis in compression testing Figure 3 shows some real-time images taken during the compression failure of the ceramic/UHMWPE armor with integral ceramic plate. As shown in Figure 3 , within 1 min after loading, the morphology around the bullet hole did not change significantly, but the cracking sound of the ceramic plate was heard, presumed to be the friction sound of residual fragments in the trajectory after bullet impact. At 3.5 min, some tearing marks were seen around the bullet hole, and the bullet hole also appeared laterally deformed; the crackling sound changed from intermittent to continuous, and the sound became clearer and clearer. It was judged that at this stage, the crack began to slowly expand outward along the circumference of the bullet hole and the overall deformation of the sample was not obvious, but the anti-cracking layer around the bullet hole appeared bulging, which was speculated to be the crack propagation of the damaged ceramic fragments around the bullet hole. After 4.5 min of loading, the bulging area and range on the surface expanded rapidly, showing a clear trend of lateral failure. During the test, small ceramic fragments broke out continuously from the bullet hole, the fracture sound was strengthened and sustained, and the brittle and short ceramic cracking sound was mixed with the continuous interlayer tearing sound. Some continuous lateral bulges were formed on the anti-cracking layer of the sample surface and extended to both side edges of the specimen, indicating that the damage had begun to expand laterally in the specimen. After 6 min of loading, the area, range, and direction of the bulges no longer changed, and it was judged that the sample had been completely damaged. (2) Failure morphology analysis The specimen after the compression failure test and its failure morphology after removing the anti-cracking cloth is shown in Figure 4 . Figure 4 a is the front of the specimen, that is, the surface of the ceramic plate impacted by the bullet. It can be seen that even if the compression load is removed, the bumps and folds formed still exist on the surface. This proves that the crack propagation led to the brittle fracture of the ceramic plate, and the bumps and folds are the manifestation of the separation of the ceramic fragments bonded to the anti-cracking cloth from the backing plate. The compression wrinkles on the surface anti-cracking layer were greatly reduced because the natural gap between the ceramic fragments was restored after the load was removed and the anti-cracking cloth layer also was roughly flat, which further confirmed that there was no debonding between the anti-cracking cloth and the ceramic plate. Figure 4 b shows the back of the specimen after unloading, that is, the back surface of the UHMWPE plate. It can be seen that the backing plate was bent along the transverse center line of the bullet hole, and the surface anti-cracking cloth was stacked laterally at the bend. This proves that the UHMWPE plate had undergone the large plastic deformation under normal pressure until it broke. The deformation and fracture caused the anti-cracking cloth to be completely debonded from the backing plate, and the layered stacks on the back surface could not be recovered even after unloading. The anti-cracking cloth in the vertical direction did not wrinkle, indicating that the shear pressure was not enough to cause obvious plastic deformation of the back plate. After the anti-cracking cloth layer wrapped around the armor sample was peeled off, the final damage of the ceramic plate and the sandwich structure can be seen as shown in Figure 4 c,d. When the anti-cracking cloth was removed, some ceramic fragments stuck to it and were taken away. Therefore, the left of Figure 4 c shows the fractured shape of the inner layer of the ceramic plate. After the ceramic fragments were carefully separated from the anti-cracking cloth and replaced on the front surface of the ceramic plate, the damage morphology is restored and is shown in the right of Figure 4 c. As can be seen from Figure 4 c, the parts near the impact point have been broken into fragments. In the right of the figure, within 100 mm around the center of the bullet hole, there are only a few radial cracks seen on the surface of the ceramic plate. In the left of the figure, the inner layer of the ceramic plate, the cracks along the circumference of the bullet hole can be classified into radial cracks, circumferential cracks, and crack cones extending to the back; the radial cracks and the circumferential cracks intersect to form a flower-like ceramic cone section; the radial crack propagation is mainly concentrated in the inner ring of ceramic cone, the circumferential crack propagation is mostly in the outer ring, and the crack distribution is roughly uniform. If the restraining effect of the anti-cracking cloth is ignored, the main causes for ballistic impact damage of the ceramic plate can be summarized as follows. At the initial stage of bullet impact, the velocity was high and the time was short, so that the area around the impact center was not sensitive to impact damage and there were few cracks (if there are, there are only a few radial cracks) on the surface of the ceramic plate because it was too late to make a failure response. As the bullet further penetrated the ceramic plate, the motion resistance increased and the bullet speed slowed down rapidly, so that the contact time with the trajectory was longer than that in the initial stage and the stress wave generated from the trajectory had enough time to propagate through the width and thickness of the target plate; therefore, a crack cone was formed. When the diameter exceeded 100 mm diameter, the crack propagation was directional, mainly extending along the horizontal direction and about ±45° direction (still intersecting in the horizontal direction); the crack propagation surface extended from the center of the bullet hole to the edge of the specimen. These transverse cracks perpendicular to the loading direction eventually led to the overall failure of the ceramic/UHMWPE armor. Figure 4 d shows the side of the ceramic/UHMWPE armor without the anti-cracking cloth, and the debonding and delamination between the two plates can be seen; a thin blade was inserted to detect the interlayer separation. It was found that the plates had been completely debonded after the compression failure test, but the largest interlayer gap was not in the fracture site of the sample but near the fracture site. This shows that in the compression test, due to the different plastic deformation degree in the backing plate and different crushing degree in the ceramic plate, the debonding on the non-fracture zone first started and became more and more serious, while on the fracture zone, the fracture first occurred and was followed by debonding, the debonding parts at the fracture being very shallow. (3) Experimental data analysis The loading force and displacement data recorded in the compression experiment were processed. The amount of collected data was too large, so it was adopted that to reduce the amount of data, 10 consecutive data were divided into a group and the median was taken. In this way, the reliability of the data was also improved. Based on the processed data, the load force/displacement curve was drawn, as shown in Figure 5 . There are three test curves in Figure 5 . They respectively correspond to the composite armor samples listed in Table 1 , which are composed of the integral ceramic plate and UHMWPE backing plate of different thicknesses. Before the compression test, all samples were impacted by a bullet at high speed. The shapes of the force/displacement curves are basically similar in Figure 5 , although the thickness of the plates constituting each sample is slightly different, which indicates that the compression failure mechanism is the same. The loading process is the most noteworthy stage, which reflects the remaining bearing capacity and failure mode of the ceramic/UHMWPE armors with ballistic damage. It can be seen from Figure 5 that during the loading stage, the overall curve changes smoothly, the slope first gradually increases and then basically remains unchanged, and the curve does not rise until the maximum compressive load. At the beginning of loading, the displacement of the sample was very small; this was at the stage of eliminating the gap, which resulted from the insufficient initial contact between the test sample and the fixture in the installation process. Under the action of the load, the various parts gradually contacted, the gaps disappeared, and the slight deformations began. When the load exceeds 10 kN, the force/displacement curve changes to a zigzag shape, which is similar to the yield phenomenon of plastic materials. In fact, it was the process of gap adjustment and the rearrangement of ceramic fragments under the action of tangential force, which was in harmony with the deformation of the UHMWPE backing plate. After that, the force/displacement curve shows a large slope without obvious fluctuations, which proves that the rearranged ceramic fragments once again played the role of the internal ceramic plate, and the deformation resistance of the whole armor improved. Before the maximum load is reached, the curve fluctuates to a zigzag shape again, indicating that the rearranged ceramic fragments began to produce new cracks and gradually separate from the backing plate, and that the backing plate also began large plastic deformation until the fracture. Briefly, the armor sample underwent the stages of ceramic fragment reorganization, plastic deformation, interlayer debonding, crack propagation, and fracture caused by compressive loads. The top of each force/displacement curve in Figure 5 shows some repeated zigzag fluctuations. This shows that after the maximum load was reached, the broken ceramic fragments were repeatedly rearranged to form a pseudo-integrity and then broken again along with interlayer crack and backing plate deformation, and the deformation resistances of the whole armor was in a state of high and low fluctuations. After such a period of time, the force/displacement curve decreases rapidly, indicating that the specimen had completely failed. From the beginning of the test, the “squeaking” sound of ceramic fragmentation was heard from the sample, which was due to the relative movement and friction between the ceramic debris remaining in the trajectory caused by bullet penetration. The ceramic debris belongs to the failed part. With the increase in load, the repeated cracking sound, composed of the brittle “squeaking” and “popping”, continued to come out. This showed that new fragmentation continued to appear in the ceramic plate during the crack propagation process. In the test, the sound of interlayer tearing was also constantly heard, especially when the maximum load was approaching, the sounds of ceramic cracking and interlayer tearing were intertwined together. The change in sound is consistent with the fluctuation in the force/displacement curve. The fragmentation of the ceramic plate absorbed a certain amount of energy and the reorganized fragments increased the deformation resistance of the sample, which was an important reason for the fluctuation of the loading force. In the fracture stage of Figure 5 , the maximum load force fluctuated many times continuously, and the sound of interlayer tearing was heard constantly at this stage of the test, indicating that the ceramic plate had been severely broken and the backing plate had been greatly deformed, the whole target gradually lost its bearing capacity. It is because of the large deformation of the backing plate and the interlayer debonding, which consume a part of the load work, that the sample has the ability to withstand such a load. It can be seen from the force/displacement curve in Figure 5 that all samples had a deformation of about 2.5~3 mm under the maximum load. Therefore, the fracture failure of ceramic/UHMWPE armors after ballistic damage is not sudden. The damaged ceramic composite armors can still work safely through deformation under a certain load. 3.2.2. Composite Armors with Spliced Ceramic Plate (1) Experimental phenomena and analysis in compression testing The morphology change in the ceramic/UHMWPE armor with the spliced ceramic plate under compressive load is shown in Figure 6 . When the compression load reached 50% of the final failure load, the intermittent crack propagation sound from the ceramic/UHMWPE armor began to be heard, but the morphology around the bullet hole in Figure 6 b seemed to show no significant change in comparison with the original state in Figure 6 a. With the increase in and continuity of fracture sound, the crack in the area around the bullet hole began to slowly expand in the lateral direction. When the load reached more than 80% of the final failure load, the obvious bulges on the anti-cracking cloth were seen from the sample in Figure 6 c, and the small ceramic particles also popped out of the bullet hole during the test. As the load continued to increase, the sound of ceramic chipping and interlayer tearing began to be heard in the sample, indicating that the damage had begun to expand inside the specimen. As shown in Figure 6 d, a continuous horizontal bulge formed on the anti-cracking cloth layer, extending to one side of the sample. The trajectory was no longer a regular circle, and there were debris bulges with different heights inside, making the surface of the trajectory very irregular. After the load reached the maximum value for a period of time, the specimen failed. (2) Failure morphology analysis Figure 7 shows the morphology of the sample after the compression failure test was performed and the anti-cracking cloth was removed. Before the compression test, the spliced ceramic/UHMWPE armor sample withstood the impact of two bullets, and two bullet holes were left on the upper and lower parts of the target plate, respectively. Figure 7 a is the front face of the sample, that is, the spliced ceramic plate. After unloading, the front surface was generally flat, the bulge formed around the bullet hole still existed, and the compression wrinkles on the surface of the anti-cracking cloth had not healed, indicating that the ceramic plate was not a completely brittle fracture and the failure was caused by the dislocation of the ceramic sheets along the joints. Even when the load was removed, the ceramic sheets still no longer returned to their original position because they had already been in a state of dislocation equilibrium. The creases on the anti-cracking cloth were caused by the protrusions on the edge of the dislocation, and there was no debonding between the anti-cracking cloth and ceramic sheets. Figure 7 b shows the back of the sample, a UHMWPE backing plate. It can be seen that the backing plate was bent along the transverse center line of the specimen (perpendicular to the direction of compressive load), rather than along the center line of the bullet hole. However, the surface anti-cracking cloth was stacked horizontally along the center lines between the two bullet holes, indicating that the UHMWPE back plate preferentially had large bending deformation between the two bullet holes, which caused the anti-cracking cloth to appear as unrecoverable local debonding even after unloading. There was no wrinkle of the anti-cracking cloth in the vertical direction, indicating that there was no sufficient shear force to cause shearing deformation and debonding of the backing plate during bending deformation. After the outer anti-cracking cloth was torn off, the final damage morphology of the spliced ceramic plate on the front and the sandwich structure on the side of the specimen could be seen, as shown in Figure 7 c,d. It took a lot of force to remove the anti-cracking cloth from the ceramic sheet because they adhered very tightly without debonding. It can be seen in the left part of Figure 7 c that a large amount of adhesive remains on the ceramic sheets. The crack on the residual adhesive indirectly reflects the damage of the spliced ceramic plate. The crack propagated radially within 100 mm of the impact center point, which was consistent with that of the integral ceramic plate. The compression failure cracks extended laterally from the center of the two impact points to both sides of the specimen because of the upper and lower compression limiting the crack propagation direction. In addition, there was a through crack between the two impact points, indicating that the impact point is the source of crack propagation and the main reason for reducing the remaining bearing capacity of the target plate. There were no visible cracks in the other parts of the adhesive surface, but some block imprints could be found, which reflected the position change of the spliced ceramic sheets. The right part of Figure 7 c shows the ceramic surface after part of the adhesive has been removed. The removed adhesive carried away some surface ceramic fragments. Some radial and circumferential cracks were seen in the spliced ceramic plate near the impact point, which were the result of the stress waves generated by the penetration movement of the warhead. The ballistic damage mechanism is the same as that of the integral ceramic plate. However, the number and density of cracks caused by ballistic damage were much less than those in the integral plate. This is mainly because the shear stress generated by the penetration of the bullet made the ceramic sheets in the spliced ceramic plate easier to move relative to each other. The relative motion absorbed a large amount of impact energy, so that the ceramic sheets were no longer broken, and only changed the position. Figure 7 d shows the side of the composite armor plate with the anti-cracking cloth removed. In order to detect the debonding and delamination between the layers, a thin blade was inserted. It can be seen that the blade was not completely inserted into the interlayer at the side, indicating that there was no interlayer debonding between the ceramic plate and the backing plate after the compression failure test. The reason should be that the size of the ceramic sheets used for splicing was small and they were dislocated and misaligned with the deformation of the backing plate under stress. The backing plate was seriously deformed and bent in an arch shape. In the test, the tearing and breaking sound of the backing plate was heard,, but no surface cracks are seen in Figure 7 d owing to the adhesive adhered on the surface and covered the fracture cracks. (3) Experimental data analysis The size of the spliced ceramic plate was large, which made it possible to study the residual strength of the ceramic/UHMWPE armor after multiple strikes. The processing method of data from the compression experiment is the same as above. The load force/displacement curves are shown in Figure 8 . They represent the compression test of the spliced ceramic/UHMWPE armor that suffered one or two bullets, respectively. The force/displacement curves in Figure 7 are generally smooth, showing a \ngradual increase in the slope until the maximal load is reached. Compared with Figure 4 , there are fewer zigzag fluctuations \nat any stage in the force/displacement curve, which is similar to the \ncompression test curve of the overall material. Such a curve shape is also \nconsistent with the failure morphology presented in Figure 6 . There are relatively few fragments on \nthe spliced ceramic plate after bullet impact, and there is almost no \ninterlayer debonding. For a long period under the initial load, the slope of \nthe curve is stable at a small value. It can be considered that the dislocation \nof the ceramic sheets caused by bullet impact did not affect the composite \ntarget plate as a whole to bear the compressive load. It can be seen from Figure 8 that both curves have a phase with an -shaped turning slope. From this stage, a very crisp and bright cracking sound “bang” \nwas heard intermittently during the test. With the increase in load, that is, \nthe rapid rising stage of the curve, the sounds of cracking, crushing, and \nfriction were continuous, which indicated that the crack was expanding inside \nsome ceramic sheets, while the other ceramic sheets were misaligned in the \ntangential direction. When the load reached the maximum value, the fracture \noccurred quickly. This is because the fracture zone caused by the ballistic \ndamage and crack propagation in the ceramic sheets had no ability to bear any \nload. The load must propagate along the unconstrained lateral direction between \nceramic sheets, which is the reason for the formation of the final fracture \nzone. In Figure 8 , the thick red solid line is the test curve of the sample fired by two bullets. There is a small zigzag wave in the rising phase of the curve, which just explains that the crack propagation caused by the two impact trajectories was not synchronous. The cracks around one trajectory first expanded and the ceramic sheets failed to load, and all the loads were quickly transferred to the other parts so that the compression damage under the influence of two trajectories merged into one at this time. It can be seen from the thick curve in Figure 8 that the slope of the rising phase after the zigzag wave is slightly reduced compared with the slope of the previous curve, indicating that the deformation resistance of the ceramic/UHMWPE armor that has been partially compressed and damaged began to decrease. When the maximum load is reached, there is a section close to a straight line at the top of the compression curve. This shows that the ceramic/UHMWPE armor after multiple blows (multiple damages) had a process of stress dispersion, transmission, and mutual offset in the state of compression failure, so that the fracture would last for a period of time and avoid sudden failure. The force/displacement curves in Figure 8 also prove that the compression failure mode and residual bearing capacity of the spliced ceramic/UHMWPE armor is different from those of the integral ceramic/UHMWPE armor, just as their ballistic damage forms are different. The ballistic impact damage of the integral ceramic/UHMWPE armor is mainly the ceramic crushing cone that resulted from the stress wave, and that of the spliced ceramic/UHMWPE armor is mainly the dislocation between the ceramic sheets. In the process of compression failure, the spliced ceramic/UHMWPE armor had no ceramic fragment reorganization, no obvious yield stage, and no interlayer debonding, while the integral ceramic/UHMWPE armor exerted its bearing capacity as a whole through self reorganization. In the same way as the integral ceramic/UHMWPE armor, the fracture of the spliced ceramic/UHMWPE armor did not happen suddenly. Therefore, the two kinds of ceramic/UHMWPE armor can maintain their working state through deformation under compression load, so as to buy time for the protection of life and property. 3.2.3. Calculation of Residual Strength and Influencing Factors After the compression test of the ceramic/UHMWPE armors that had suffered bullet impact, the residual compressive strength of each sample can be calculated. The calculation formula is shown in Equation (1).\n (1) R CAI = P max A \nwhere: R CAI is the residual compressive strength (MPa); P max is the maximum compressive force borne by the sample before failure (N); A is the cross-sectional area of the sample (mm 2 ). The calculation results are shown in Table 1 . The relationship between compressive strength and ceramic plate thickness is shown in Figure 9 . By comparing the data in Table 1 , it is known that the greater the plate thickness is, the greater the residual strength is, whether the ceramic/UHMWPE armor is with an integral ceramic plate or with a spliced ceramic plate (similarly hit by one bullet or two bullets). However, when the plate thickness is increased from 10 mm to 12 mm, the residual strength does not change much, which indicates that there should be an appropriate value between the thickness of the ceramic plate and its protection ability when preparing a composite armor, and it can be selected and designed according to the protection requirements. After being hit similarly by a bullet, the residual strength of the spliced ceramic/UHMWPE armor is significantly greater, which is almost twice that of the integral ceramic/UHMWPE armor, indicating that the spliced ceramic/UHMWPE armor has higher protection ability after bullet damage. After being hit by two bullets, the residual strength of the spliced ceramic/UHMWPE armor reduced a lot, but such a small residual strength still meets the design load (>0.004782 Mpa) requirements in CCAR-25 [ 27 ]. 3.3. Future Research Directions The main task of this research is to experiment, analyze, and discuss the experimental phenomena, so as to reveal the projectile penetration mechanism of ceramic/UHMWPE armors and the residual bearing capacity after damage. However, the test cost is high and the number of tests is limited. In the future, it is necessary to numerically simulate the test phenomena and establish the model for optimizing the armor structure and predicting failure. The initial tests provide a basis for verifying the fidelity of the numerical model and material model parameters. The combination of the experiment and numerical simulation has been proved to be very effective in studying the failure mechanism of the armor plate [ 28 ]. In fact, when the projectile strikes the ceramic armor, part of the impact energy is converted into heat energy, which increases the local temperature of the armor material, resulting in the change of material properties. It is difficult to observe and capture these changes during the test, but this is a problem that must be paid attention to, especially when establishing the numerical model. The flow stress model of a projectile hitting hard armor plate has been established, which is a cumulative damage fracture model considering loading history [ 28 ]. When the impact equivalent stress borne by the target plate is calculated, not only the experimental loading parameters but also the influence of thermal softening are considered, including the experimental ambient temperature T , room temperature T r , and the melting point temperature T m of the material, which are normalized to T H = T − T r T m − T r . From the normalized temperature equation, it can be seen that the operating environment of the armor material has a certain impact on the impact stress. At extreme high and low temperatures, penetration results may show differently from those in laboratory tests. The higher the melting point of the target material, the less the impact of the working environment temperature on the penetration results, which further explains the reason for choosing high temperature resistant materials as bulletproof targets [ 1 , 2 , 3 ]. Previous studies have also proved that the impact energy absorption capacity of polyphylene, polypropylene, and their composites are affected by impact temperature, impact velocity, and strain rate [ 29 , 30 ]. These research results will provide guidance for the establishment of a numerical analysis model in the next step."
} | 10,600 |
34311573 | PMC8406198 | pmc | 4,223 | {
"abstract": "ABSTRACT Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2 PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe 2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe 2+ , in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/ Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism.",
"introduction": "INTRODUCTION Iron (Fe) oxidation occurs in virtually all near-surface environments, producing highly reactive iron oxyhydroxides that often control the fate of carbon, phosphorus, and other metals ( 1 ). It is often assumed that abiotic reactions are the primary mechanisms of iron oxidation, particularly at near-neutral pH. However, iron-oxidizing microbes are increasingly observed in a wide range of environments, especially dark, neutral pH environments such as aquifers, soils, sediments, hydrothermal vents, and water treatment systems ( 2 – 7 ). Neutrophilic iron oxidizers thrive at anoxic-oxic interfaces where they can outcompete abiotic iron oxidation rates at low oxygen concentrations ( 8 ). Iron-oxidizing microbes can grow by coupling iron oxidation to the reduction of oxygen or nitrate, using this energy to fuel carbon fixation and biomass production ( 2 , 9 , 10 ), but in many iron-replete environments, we lack a clear understanding of the extent of microbial iron oxidation. To address this, we need to confidently identify iron oxidation mechanisms. Yet, unlike other major microbial metabolisms, we have relatively incomplete knowledge of iron oxidation pathways ( 11 – 13 ). Rising interest in iron-oxidizing microbes has resulted in a surge of iron oxidizer sequencing, including isolate genomes, single cell genomes, metagenomes, and metatranscriptomes ( 5 , 7 , 9 , 14 – 20 ), enabling us to search for the genes involved in microbial iron oxidation using genome mining. All sequenced genomes of the known neutrophilic chemolithoautotrophic iron-oxidizing bacteria (FeOB), the marine Zetaproteobacteria ( Mariprofundus spp., Ghiorsea spp.) and freshwater Gallionellaceae ( Gallionella spp., Sideroxydans spp., and Ferriphaselus spp.), have homologs to cyc2 ( 7 , 21 – 25 ), which encodes an iron-oxidizing outer membrane cytochrome first characterized in Acidithiobacillus ferrooxidans ( 26 , 27 ). Many of these FeOB are obligate iron oxidizers that lack other apparent iron oxidase candidates. A second potential iron oxidase gene, mtoA , is found in a few Gallionellaceae and Thiomonas genomes ( 21 , 28 , 29 ), and functional and genetic information supports the role of MtoA and its homolog PioA in iron oxidation ( 30 – 32 ). However, relatively few FeOB genomes contain mtoA , and pioA is limited to phototrophic organisms, suggesting that Cyc2 is potentially a more widespread iron oxidase. Recently, McAllister et al. presented the phylogeny of 634 unique Cyc2 homologs ( 7 ), which resolved into three distinct clusters. Two of the clusters each contain a Cyc2 homolog with verified iron-oxidizing activity— A. ferrooxidans Cyc2 ( 27 ) in Cluster 2 and Leptospirillum sp. strain Cyt 572 ( 33 ) in Cluster 3. Both of these organisms are acidophilic iron oxidizers. Cluster 1 consists largely of the well-known neutrophilic iron oxidizers, including the Zetaproteobacteria , Gallionellaceae , and iron-oxidizing Chlorobium spp. This cluster is well supported, and these sequences are among the closest homologs to one another despite the taxonomic distance between these organisms ( 7 ). A common iron oxidation pathway for both neutrophiles and acidophiles might not be expected, due to the drastically different redox potential of Fe(II)/Fe(III) at acidic versus neutral pH (770 mV at pH 2 versus 0 ± 100 mV at pH 7 [ 11 , 34 – 36 ]), but a conserved protein structure could suggest a shared function. To be more confident that Cyc2 is an iron oxidase in a wide range of iron oxidizers, we need biochemical verification of Cyc2 activity from neutrophilic chemolithotrophic FeOB. Our main objective was to test for iron-oxidizing activity by Cyc2 from the well-supported Cluster 1 containing neutrophilic FeOB. We chose the Cluster 1 Cyc2 from Mariprofundus ferrooxydans PV-1, an obligate iron oxidizer, with cyc2 as the only identified iron oxidase gene candidate in its genome ( 7 , 37 – 39 ). Environmental metatranscriptomics of marine iron-oxidizing microbial mats dominated by Zetaproteobacteria , including Mariprofundus spp. found high expression of cyc2 , along with evidence that cyc2 expression may be regulated in response to Fe(II), suggesting utility for cyc2 in an iron-oxidizing environment ( 7 , 10 ). Proteomics of M. ferrooxydans PV-1 showed that Cyc2 was expressed, and a membrane complex containing Cyc2 possessed ferrocyanide oxidation activity ( 39 ). While the A. ferrooxidans Cyc2 characterization was performed in the native organism ( 27 ), neutrophilic FeOB are challenging to grow in quantities sufficient for protein assays, so we took a heterologous expression approach. We first performed structure-function predictions to inform the design of the expression constructs, which we then expressed in Escherichia coli . To test the iron oxidase function, we expressed Cyc2 from M. ferrooxydans PV-1 (Cyc2 PV-1 ) under native conditions and performed iron oxidation assays and redox potential measurements of Cyc2 PV-1 enriched by purification chromatography. We integrate structural, functional, and phylogenetic insights to explore the function of a wide range of Cyc2 homologs, including strategies for correctly identifying Cyc2 homologs and interpreting Cyc2 iron-oxidizing function.",
"discussion": "RESULTS AND DISCUSSION Cyc2 is a predicted outer membrane cytochrome fused to a porin. We first performed a primary and secondary structure analysis to better understand the functional parts of Cyc2 PV-1 prior to expression. Cyc2 starts with a signal peptide, predicted by SignalP ( 40 ) and has a single -CXXCH- heme-binding motif ( 41 ), placing this cytochrome in the c -type family. Fused to the cytochrome domain is a beta-barrel porin, based on the presence of 16 beta strands predicted by PSIPRED ( 42 , 43 ) (see Fig. S1 in the supplemental material) and homology matching by HHpred ( 44 , 45 ). The porin-encoding segment has low sequence homology but high structural homology to the outer membrane phosphate-selective porins OprO and OprP (PDB structures 4RJW and 2O4V [ 46 , 47 ]), based on structural homology predictions by I-TASSER, MODELLER, and Phyre2 ( 48 – 51 ) ( Fig. 1 ). We performed HHpred structural predictions for a diverse set of Cyc2 sequences from McAllister et al. ( 7 ), and all analyzed sequences matched to the same tertiary structure. Thus, all Cyc2 sequences are predicted to have the same structure: a 16-stranded outer membrane porin with an N-terminal cytochrome domain ( Fig. 1 ). FIG 1 I-TASSER ( 49 ) model of Cyc2 PV-1 . Cyc2 is predicted to be a 16-stranded porin with a fused N-terminal cytochrome domain. The Cyc2 cytochrome (cyt) (purple oval) is connected to the N-terminal (blue strand) end of the porin. The view on the right is rotated 90 ° toward the viewer to show the internal pore of the porin but does not contain the cytochrome. 10.1128/mBio.01074-21.1 FIG S1 PSIPRED prediction of secondary structure in Cyc2 PV-1 , the Cyc2 sequence from M. ferrooxydans PV-1 (D. W. A. Buchan, D. T. Jones, Nucleic Acids Res 47:W402–W407, 2019, https://doi.org/10.1093/nar/gkz297 ). Download FIG S1, PDF file, 0.2 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . The combination of signal peptide and beta-barrel structure suggests Cyc2 is localized to the outer membrane, as expected for a porin ( 52 ). This location is consistent with previous observations that iron oxidation occurs at the cell surface, preventing internal iron oxyhydroxide encrustation ( 53 , 54 ). Porins typically possess short periplasmic turns and longer extracellular loops and have both the N and C termini in the periplasmic space ( 55 ). This standard orientation applied to Cyc2 would suggest that the cytochrome domain of Cyc2 resides on the periplasmic side of the barrel, likely as a plug at the opening of the beta-barrel pore. Strategy for expression of fused cytochrome-porin. Overexpression strategies for porin proteins typically include directing expression to the cytoplasm where they form inclusion bodies that can be purified, and the porin is subsequently refolded ( 56 ). However, c -type cytochromes must be matured in the oxidizing environment of the periplasm to ensure covalent attachment of the heme ( 57 ). Thus, to ensure proper folding and localization to the outer membrane, our expression strategies were restricted to native conditions, at the expense of yield. To maintain these functional parts during expression in E. coli C41, we synthesized a codon-optimized gene construct, and replaced the PV-1 signal peptide with the signal peptide from E. coli OmpA ( 56 ). We replaced the signal peptide to ensure E. coli directed the nascent polypeptide for translocation to the periplasm, where it could then interact with outer membrane insertion machinery ( 55 ). To assist with detection, we placed a Strep tag II at the C terminus where it would likely not affect cytochrome maturation ( Fig. S2A ). For purification, we additionally placed a linker, tobacco etch virus (TEV) protease cleavage site, and octahistidine tag (His tag) following the Strep tag II ( Fig. 2A ). In addition, we tested independent expression of the cytochrome domain and the porin domain, and while the porin was expressed, the cytochrome was not (data not shown), suggesting contacts within the full structure play a role in its stability. FIG 2 Constructs and expression of Cyc2 PV-1 . The position of Cyc2 PV-1 is marked with a red arrowhead. The protein ladder is shown in lanes M (Spectra Broad Range on heme and Coomassie blue, WesternC on α- Strep tag Western blots), and relevant band sizes are labeled in kilodaltons. (A) Schematic of gene construct for expression in E. coli . (B) Representative stained SDS polyacrylamide gels showing Cyc2 PV-1 expression in E. coli that was uninduced (lane 1), induced (lane 2), and lysed and induced (lane 3). Smaller bands visible on the Strep tag Western blots are nonspecific. (C) Heme-stained gel of fractions during His tag purification. After elution, Cyc2 PV-1 migrates in a high-molecular-weight complex. (D) Stained SDS polyacrylamide gels showing Cyc2 PV-1 enrichment fraction after His tag purification (lane 4), after cleavage of His tag (lane 5), and after concentration for assays (lane 6). Uncropped gels are shown in Fig. S2C in the supplemental material. 10.1128/mBio.01074-21.2 FIG S2 Constructs and expression of Cyc2 PV-1 . The position of Cyc2 PV-1 is marked with a red arrowhead. The protein ladder is shown in lane M (Spectra Broad Range on heme and Coomassie blue, WesternC on α- Strep tag Western blot), and relevant band sizes are labeled in kilodaltons. (A) Schematic of gene construct for expression and representative stained SDS-PAGE gels showing Cyc2 PV-1 expression in E. coli that was uninduced (lane 1), induced (lane 2), and lysed and induced (lane 3). Smaller bands visible on Strep tag Western blots are nonspecific. (B) Heme-stained gel of fractions during His tag purification. Cyc2 PV-1 migrates at its expected molecular weight in the diluted total membranes (input) and flowthrough (FT). After elution, Cyc2 PV-1 migrates in a high-molecular-weight complex (lane 1, 300 μM imidazole; lane 2, after dialysis to remove imidazole). After TEV protease cleavage of the His tag, Cyc2 PV-1 does not interact with the Ni-NTA column (lane 3, flowthrough; lane 4, imidazole elution). (C) Uncropped gel corresponding to Fig. 2D . See lane labels to the right of the image. Download FIG S2, PDF file, 0.2 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Cyc2 PV-1 was produced by heterologous expression in E. coli . We confirmed production of Cyc2 PV-1 in total E. coli lysate by immunoblotting with antibodies specific to the C-terminal Strep tag II, as Cyc2 PV-1 could not be identified solely by Coomassie blue staining (construct with both Strep and His tag [ Fig. 2B ]; construct with Strep tag only [ Fig. S2A ]). We confirmed that this same protein contained heme. We were unsuccessful at purification using only the Strep tag, but the His tag could successfully be used for enrichment of Cyc2 PV-1 from E. coli extracts. Prior to enrichment, Cyc2 PV-1 ran at its apparent molecular weight and was the only heme-containing protein present. After enrichment, Cyc2 PV-1 no longer ran true to size and instead appeared as a high-molecular-weight band, possibly due to the purification conditions (high salt, high imidazole, and increased protein concentration) ( Fig. 2C and D ; Fig. S2B ). We confirmed Cyc2 PV-1 is an outer membrane protein by using a sucrose gradient and ultracentrifugation to separate membrane components ( 58 , 59 ). Cyc2 PV-1 was found in the outer membrane fraction, in agreement with the predictions from the bioinformatic analysis ( Fig. 3A ). We isolated the band corresponding to Cyc2 PV-1 from the outer membrane fraction on a heme-stained sodium dodecyl sulfate (SDS) polyacrylamide gel. Tandem mass spectrometry analysis of this band confirmed its identity as Cyc2 PV-1 , as nearly 60% of the protein sequence was detected, including regions of the cytochrome domain, the porin domain, and the Strep tag II ( Fig. 3B ; see also Table S1 in the supplemental material). FIG 3 Cyc2 PV-1 is located in the outer membrane. (A) Heme-stained SDS polyacrylamide gel of fractionated E. coli (CP1 and CP2 are cytoplasmic proteins; IM, inner membranes; OM, outer membranes; M, Spectra Broad Range protein ladder with relevant bands in kilodaltons). Cyc2 PV-1 was found only in the outer membrane fraction (OM), and no other heme-containing proteins were seen in the OM. A Coomassie blue-stained gel is shown in Fig. S3 . (B) Highlighted yellow peptides observed in tandem MS/MS confirmed the identity of the OM heme-stained band as Cyc2 PV-1 , with nearly 60% of the protein detected. The signal peptide is crossed out, as it is not present in the mature protein. 10.1128/mBio.01074-21.9 TABLE S1 Unique peptides detected by tandem MS/MS that matched to Cyc2 PV-1 with 99% confidence. Download Table S1, PDF file, 0.1 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.01074-21.3 FIG S3 Uncropped gels. (A) Coomassie blue-stained gel corresponding to Fig. 3A . Lanes: CP1 and CP2, cytoplasmic proteins; IM, inner membranes; OM, outer membranes; M, Spectra Broad Range protein ladder with the relevant bands in kilodaltons. (B) Gels corresponding to samples in Fig. 4 . A red arrowhead indicates full-length Cyc2 PV-1 , and a blue arrowhead indicates porin only. Lanes: 1, empty vector; 2, porin lysed supernatant; 3, porin ultracentrifuged supernatant; 4, porin total membranes; 5, porin cytoplasmic proteins; 6, porin inner membranes; 7, porin outer membranes; 8, Spectra Broad Range or WesternC protein ladder; 9, Cyc2 PV-1 lysed supernatant; 10, Cyc2 PV-1 ultracentrifuged supernatant; 11, Cyc2 PV-1 total membranes; 12, Cyc2 PV-1 cytoplasmic proteins; 13, Cyc2 PV-1 inner membranes; 14, Cyc2 PV-1 outer membranes. Download FIG S3, PDF file, 0.1 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Cyc2 PV-1 has a distinct heme spectrum. We isolated inner and outer membranes from both E. coli expressing full-length Cyc2 PV-1 and E. coli expressing only the porin domain of Cyc2 PV-1 . The UV-visible (UV-Vis) absorbance spectra of these two E. coli membrane fractions were compared to identify the heme signal from Cyc2 PV-1 ( Fig. 4 ). The inner membranes of E. coli expressing either Cyc2 PV-1 or porin domain only were virtually identical when analyzed by UV-Vis spectroscopy, showing a heme Soret peak at 415 nm ( Fig. 4 , dashed black and orange spectra). These peaks represent E. coli ’s native heme-containing proteins. In contrast, there are no native outer membrane heme proteins in E. coli ( 60 ). Indeed, only the outer membranes from E. coli expressing full-length Cyc2 PV-1 had heme ( Fig. 4 , solid orange spectrum), with a Soret peak at 410 nm. Thus, the Soret peak at 410 nm is indicative of Cyc2 PV-1 in our system and can be used to spectroscopically detect Cyc2 PV-1 . FIG 4 Cyc2 PV-1 has a distinctive heme Soret peak of 410 nm. UV-Vis spectra of inner membranes (IM) and outer membranes (OM) obtained from a sucrose gradient of E. coli expressing either full-length Cyc2 PV-1 or only the porin domain of Cyc2 PV-1 . Cyc2 PV-1 OM had heme (orange), while porin OM did not (black), and the heme signal was distinct from other heme proteins in E. coli (IM, dashed black and orange). Heme-stained and Coomassie blue-stained SDS polyacrylamide gel and Strep tag Western blot are shown in Fig. S3 . Cyc2 PV-1 is an iron oxidase. We assayed the iron oxidation capacity of enriched Cyc2 PV-1 by UV-Vis spectroscopy under anaerobic conditions after removal of the His tag by TEV protease cleavage. While Cyc2 PV-1 is not pure, it is the only heme-containing protein detected, and the UV-Vis methods utilized here rely only on the heme spectra. Importantly, the heme spectrum of enriched Cyc2 PV-1 is identical to the heme spectrum of outer membranes from E. coli expressing Cyc2 PV-1 , where Cyc2 PV-1 was established as the only heme-containing protein ( Fig. 4 ). Cyc2 PV-1 could be reduced with sodium dithionite ( Fig. 5A ), demonstrated by the shift of the Soret peak from 410 nm to 427 nm and the appearance of α and β peaks at 560 nm and 530 nm, respectively. Addition of an oxidizing agent to enriched Cyc2 PV-1 caused no changes, indicating that Cyc2 PV-1 was in the oxidized state as purified ( Fig. 5A ). These assays demonstrated Cyc2 PV-1 was redox active, and so we tested its capacity to oxidize Fe(II). FIG 5 Cyc2 PV-1 is redox active and is an iron oxidase. UV-Vis spectra of Cyc2 PV-1 after enrichment chromatography and cleavage of the His tag. (A) Cyc2 PV-1 as purified was oxidized (black). Addition of potassium hexacyanoferrate(III) showed no changes to the heme spectra, confirming the oxidized state (gray). Sodium dithionite reduced Cyc2 PV-1 (red), as evidenced by the shift in the Soret peak and the appearance of the alpha and beta peaks (black arrows). (B) Cyc2 PV-1 as purified (black). Fe(II) reduced Cyc2 PV-1 (red), shown by the shift in the Soret peak and the appearance of the same alpha and beta peaks as the dithionite-reduced spectra. To perform the iron oxidation assay, we used Fe(II) with citrate to chelate the Fe(III) product, preventing formation of iron oxyhydroxide precipitates that would interfere with the UV-Vis assay. Citrate is a weak Fe(II) ligand (stability constant log K = 3.20; https://www.nist.gov/srd/nist46 ), readily releasing Fe 2+ , and has been found not to interfere with iron oxidation in FeOB ( 61 ). We calculated that ∼20% of the Fe(II) is Fe 2+ in our assay solution ( Table S2 ) ( 62 ), so Fe 2+ is available as a substrate. Reaction between Fe(II) and Cyc2 PV-1 showed the same shift of the Soret peak and appearance of the same alpha and beta peaks as reduction by sodium dithionite ( Fig. 5B ). These spectral changes show Cyc2 PV-1 can accept electrons from Fe(II), demonstrating that Cyc2 PV-1 can function as an iron oxidase. 10.1128/mBio.01074-21.10 TABLE S2 Relevant Fe(II) and citrate speciation in the iron oxidase assay buffer from Visual MINTEQ calculation ( https://vminteq.lwr.kth.se/ ). Download Table S2, PDF file, 0.1 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Cyc2 PV-1 has a redox potential of 208 ± 20 mV. To determine the redox potential of Cyc2 PV-1 , we used a modified Massey method protocol developed for low-yield heme proteins ( 63 – 65 ). Cyc2 PV-1 was titrated against 2,6-dichlorophenolindophenol (DCPIP) with a known redox potential of +217 mV ( 64 ). By plotting the Nernst equation-transformed ratios of oxidized and reduced forms of both DCPIP and Cyc2 PV-1 from four independent experiments ( Fig. S4 ), we calculated the redox potential of Cyc2 PV-1 as 208 ± 20 mV ( Fig. 6 ). FIG 6 Cyc2 PV-1 redox potential measurements shown by representative UV-Vis spectra and data plotting. (A) Spectroscopic changes observed during the determination of the reduction potential of Cyc2 PV-1 using the dye DCPIP. UV-Vis spectra were recorded every 15 s during a xanthine oxidase-catalyzed reductive titration with xanthine, DCPIP, and Cyc2 PV-1 . (B) The ratios of the reduced and oxidized forms of Cyc2 PV-1 and DCPIP were plotted and used to determine the redox potential of Cyc2 PV-1 . 10.1128/mBio.01074-21.4 FIG S4 Four independent redox titration reactions with Cyc2 PV-1 and DCPIP. Download FIG S4, PDF file, 0.04 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Our results show a redox interaction between Cyc2 PV-1 and Fe(II) ( Fig. 5 ), and the calculated redox potential of Cyc2 PV-1 ( Fig. 6 ) puts it between the redox potentials of Fe(III)/Fe(II) (pH 7) and O 2 /H 2 O ( Fig. 7 ), as would be expected for a neutrophilic iron oxidizer. In contrast, the redox potential for Cyc2 from A. ferrooxidans was measured at 560 mV ( 27 ) which is consistent with the higher Fe(III)/Fe(II) redox potential at low pH. These differences suggest Cyc2 has evolved to have appropriate redox potentials for respective FeOB environments. FIG 7 Schematic of reduction potentials showing how Cyc2 PV-1 fits into the neutrophilic electron transport pathway. Boxes represent the range of reported values: for both the Fe(III)/Fe(II) couple (Fe 2+ /ferrihydrite couple, as predicted from FeOB mineralogy) and menaquinones and ubiquinones, this ranges from −110 to +110 mV. Much remains unknown in the “downhill” electron transport to the terminal electron acceptor, oxygen, but likely involves other cytochromes ( 11 , 34 ). Differentiating the structure of Cyc2 from other iron oxidoreductases. In total, these results bolster the evidence thus far that Cyc2 is an outer membrane iron oxidase. While Cyc2 is unique in that the cytochrome and porin are fused, its overall structure is reminiscent of other porin cytochrome complexes that play roles in iron cycling, particularly MtrCAB/OmcA and MtoAB/PioAB ( 30 – 32 , 66 – 68 ). These complexes include a 26-strand beta-barrel that accommodates insertion of a decaheme cytochrome, which spans the outer membrane and may contact other extracellular decaheme proteins to conduct extracellular electron transfer ( Fig. 8 ). In contrast, Cyc2 is predicted to have a single heme and a smaller barrel (16 strands). For organisms that eke out a living from iron oxidation, the single heme and smaller size of Cyc2 mean it requires fewer resources to produce, making it a streamlined alternative to larger porin cytochrome complexes ( 69 ). FIG 8 Cyc2 is different than other iron oxidoreductases. The MtrCAB complex is a 26-stranded porin with hemes spanning a range of 185 Å ( 67 ). The PioAB/MtoAB complex is likely similarly sized to MtrAB, with hemes spanning up to ∼100 Å based on modeling ( 32 ). Cyc2 is a smaller porin of 16 strands and possesses only a single heme. Due to its smaller size and placement of single heme, Fe 2+ would have to enter the pore of Cyc2. If the pore size of Cyc2 is similar to the structurally homologous phosphate porins, the internal diameter is expected to be ∼3.5 Å ( 46 ). A model of the Cyc2 PV-1 cytochrome domain is ∼20 × 20 × 10 Å ( Fig. S5 ), which would fit as a plug within the periplasmic opening of the barrel, but not span the outer membrane through the porin, nor fully reside within the middle of the pore. Given our structural predictions, the cytochrome would be on the periplasmic side of Cyc2, which would allow transfer of electrons to a periplasmic component of the electron transport chain. While long-range electron transport is possible, the rate of electron transport decreases exponentially with distance ( 70 ). The hemes within MtrA are 3.9 to 6.5 Å apart ( 67 ), and similar configurations are found in other cytochromes as well, suggesting this distance is optimal for efficient electron transport. In our model, Fe 2+ would need to enter the Cyc2 pore to some extent, possibly with the help of a chaperone or ligand that could also escort Fe 3+ from the pore before formation of Fe(III) oxyhydroxides that would detrimentally encrust the protein. The requirement for iron to enter the pore would suggest that Cyc2 is an oxidase of dissolved Fe 2+ , distinguishing it from a multiheme iron oxidase like MtoA that could directly conduct electrons from a solid surface. 10.1128/mBio.01074-21.5 FIG S5 Three views of the modeled cytochrome domain of Cyc2 PV-1 . The view on the right is rotated 90 ° away from the viewer compared to the view in the center. Hydrophobic residues are colored gray, and polar residues are colored red. Heme (not pictured) is covalently attached to cysteine residues (yellow) and coordinated by histidine (blue). The model was generated using MODELLER (B. Webb, A. Sali, Curr Protoc Bioinformatics 54:5.6.1−5.6.37, 2016, https://doi.org/10.1002/cpbi.3 ). Download FIG S5, PDF file, 0.1 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Recognizing cyc2 /Cyc2 in other organisms. In the short time since the first comprehensive Cyc2 phylogenetic tree was published ( 7 ), many more cyc2 homologs have been sequenced. To explore how these new sequences fit into our understanding of Cyc2 phylogeny, we updated the Cyc2 tree with sequences from databases that met the criteria of our in-house pipeline—specifically a minimum length (365 amino acids), presence of a heme-binding site, and low blastp similarity cutoff (1E−5). Even with the substantial increase in Cyc2 homologs, the topology of the tree remained the same, with strong support for three main clusters of Cyc2 sequences ( Fig. 9 ; see figshare File 2 at https://doi.org/10.6084/m9.figshare.c.5390285 ). FIG 9 Updated Cyc2 maximum likelihood phylogenetic tree (1,593 sequences total). Sequences come from isolates, enrichments, single amplified genomes, and metagenome-assembled genomes, with few replicates. The three Cyc2 clusters continue to be supported (79% bootstrap support) and are confirmed with HMMs from FeGenie. Functionally characterized Cyc2 are shown with asterisks, with one from each cluster. Known neutrophilic Fe(II)-oxidizing clades are labeled with the number of distinct isolates represented by each. The full rectangular version of the tree with leaf labels is shown in figshare File 2 at https://doi.org/10.6084/m9.figshare.c.5390285 . We have historically relied on our in-house pipeline for identifying Cyc2 homologs because the Cyc2 sequence is dominated by a porin domain that characteristically features low amino acid conservation ( 71 ). In contrast, the short cytochrome domain is the most conserved region ( Fig. S6 and S7 ), and a consensus sequence derived from all homologs includes an AXPXFAR [Q/K][T/Y] motif located 5 amino acids upstream of the CXXCH heme-binding site, and a PXL motif 4 amino acids downstream of the CXXCH ( Fig. S8 ). The PXL motif can be found in many other cytochromes, such as MtoD ( 72 ) and Cyc1 in A. ferrooxidans (CYC41 in reference 73 ); the proline and lysine appear to help stabilize the heme ( 73 ). In contrast, the AXPXFAR [Q/K][T/Y] motif is unique to Cyc2, and therefore can be used to identify Cyc2-like cytochromes. 10.1128/mBio.01074-21.6 FIG S6 Full alignment of Cyc2 from representative neutrophilic and acidophilic FeOB. The orange line indicates the conserved cytochrome region. Download FIG S6, PDF file, 1.9 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.01074-21.7 FIG S7 Histograms of pairwise amino acid identity of the full-length Cyc2 sequences (A), porin portion (B), and cytochrome portion ( n = 156) (C). The cytochrome portion is more highly conserved than the porin. (D) Amino acid identities (AAI) of full-length Cyc2 sequences from FeOB and Tenderia electrophaga . AAI to biochemically characterized Cyc2 are shown in bold type. Note that organisms from Cluster 1, e.g., neutrophilic FeOB Zetaproteobacteria , Gallionellaceae , and Chlorobi , are most similar to one another. Download FIG S7, PDF file, 0.2 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.01074-21.8 FIG S8 Comparison of motifs found in the conserved cytochrome domain of Cyc2. The sequence logo labeled “All” is built from 1,593 homologs. Each of the cluster logos are built from all sequences in each cluster (334 in Cluster 1, 858 in Cluster 2, and 401 in Cluster 3). Download FIG S8, PDF file, 0.5 MB . Copyright © 2021 Keffer et al. 2021 Keffer et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Recently, we developed hidden Markov models (HMMs) to identify Cyc2 sequences. These HMMs are part of the FeGenie program ( 74 ), which identifies and assigns putative Cyc2 homologs to one of the three phylogenetic clusters, depending on which HMM is the highest-scoring match. The HMMs were built from representative sequences of the phylogenetic tree presented by McAllister et al. ( 7 ), and each phylogenetic cluster is represented by a unique HMM. An alignment of sequences from each cluster show some differences in the consensus motif between clusters ( Fig. S8 ). The FeGenie algorithm confirms the presence of a heme-binding motif and excludes sequences shorter than 365 amino acids, but users should confirm the presence of a porin in FeGenie-identified Cyc2 homologs using secondary structure prediction software (e.g., HHPRED). We confirmed the FeGenie Cyc2 HMM cluster classification matched the location of the sequences in the updated Cyc2 tree, with only one exception (figshare File 2 at https://doi.org/10.6084/m9.figshare.c.5390285 ). The Cyc2 HMMs have been validated and are the recommended tool for finding new Cyc2 homologs ( 74 ). Interpreting Cyc2 sequences: toward the discovery of new iron oxidizers. We now have biochemical evidence for iron oxidation function by three diverse Cyc2 representatives that each fall within one of the three main clusters. It is tempting to now attribute iron oxidation function to the various Cyc2 homologs found in organisms across the tree ( Fig. 9 ), as well as new homologs found using the HMM-based strategy above. However, the degree of confidence depends heavily on context, particularly how closely related the homolog is to a characterized Cyc2. New sequences can be added to the Cyc2 phylogenetic tree using the full alignment file (figshare Files 3 and 4 at https://doi.org/10.6084/m9.figshare.c.5390285 ). In some cases, the new sequence may fall within a well-supported clade of known FeOB with good evidence for Cyc2 function, which will give confidence in its predicted role in iron oxidation. However, the many long branches on the Cyc2 tree indicate a large degree of divergence, notably in Cluster 3. The functionally characterized Leptospirillum Cyt 572 has an especially long branch, and it also has an unusual heme prosthetic group ( 75 ), both of which could mean that Cyt 572 is not necessarily representative of Cluster 3. In all, the function of cyc2 homologs within Cluster 3 may be considered most uncertain, requiring further verification. In contrast, Cluster 1 has shorter branches, indicating that the sequences are more closely related to one another and to the biochemically tested Cyc2 PV-1 . In fact, many of the sequences (∼55%) within this cluster are from well-characterized neutrophilic FeOB, i.e., the Gallionellaceae , Zetaproteobacteria , or Chlorobi , including all known iron-oxidizing isolates of those taxa. In addition, previous work has helped build a case for prevalent Cluster 1 Cyc2 function. Cluster 1 FeOB cyc2 are highly expressed in iron-oxidizing environments, including Gallionellaceae cyc2 in the Rifle alluvial aquifer ( 5 ) and Zetaproteobacteria cyc2 in marine iron mats in three hydrothermal systems ( 7 ). This hints at their importance in iron oxidation, which is further supported by expression that corresponds to iron-oxidizing conditions stimulated in microcosms ( 7 ). Building on this, here we show that a Cluster 1 Cyc2 oxidizes Fe(II) and has an appropriate redox potential. Altogether, this gives a higher degree of confidence in generalizing the iron oxidation function across Cluster 1 Cyc2. The Cyc2 phylogenetic tree shows that Cyc2 sequences are very diverse, with many homologs that are distantly related to Cyc2 PV-1 , A. ferrooxidans Cyc2, and Cyt 572 , notably much of Clusters 2 and 3. Thus, there is still much work to be done to verify Cyc2 function in both known FeOB and organisms not known to oxidize iron. It is certainly possible that Cyc2 has additional functions, so its role in iron oxidation should be tested by genetic methods, though these have proven to be challenging for FeOB. However, the vast majority of cyc2 /Cyc2 sequences come from genomes of uncultured organisms. In these cases, how can we test the function? We recently discovered Cluster 1 cyc2 sequences in metagenome-assembled genomes of “ Candidatus Ferristratum” spp. from the DTB120 phylum ( Fig. 9 ), found in deeper portions of hydrothermal iron microbial mats inhabited by well-characterized Zetaproteobacteria FeOB. To test whether the new “ Ca. Ferristratum” cyc2 is connected to iron oxidation, we added Fe(II) to iron mat sample microcosms and analyzed a time course of transcriptomes. The expression of cyc2 and the nitrate reduction gene narG from “ Ca. Ferristratum” increased concurrently in response to Fe(II) addition, suggesting this group of organisms couples iron oxidation to nitrate reduction ( 10 ). This approach can guide the exploration of the diversity, distribution, and roles of iron-oxidizing bacteria in environmental systems. If many other Cyc2 homologs prove to be iron oxidases, microbial iron oxidation may be more widespread than we currently recognize."
} | 9,321 |
37752304 | PMC10923983 | pmc | 4,225 | {
"abstract": "Ant species exhibit behavioural commonalities when solving navigational challenges for successful orientation and to reach goal locations. These behaviours rely on a shared toolbox of navigational strategies that guide individuals under an array of motivational contexts. The mechanisms that support these behaviours, however, are tuned to each species’ habitat and ecology with some exhibiting unique navigational behaviours. This leads to clear differences in how ant navigators rely on this shared toolbox to reach goals. Species with hybrid foraging structures, which navigate partially upon a pheromone-marked column, express distinct differences in their toolbox, compared to solitary foragers. Here, we explore the navigational abilities of the Western Thatching ant ( Formica \n obscuripes ), a hybrid foraging species whose navigational mechanisms have not been studied. We characterise their reliance on both the visual panorama and a path integrator for orientation, with the pheromone’s presence acting as a non-directional reassurance cue, promoting continued orientation based on other strategies. This species also displays backtracking behaviour, which occurs with a combination of unfamiliar terrestrial cues and the absence of the pheromone, thus operating based upon a combination of the individual mechanisms observed in solitarily and socially foraging species. We also characterise a new form of goalless orientation in these ants, an initial retreating behaviour that is modulated by the forager’s path integration system. The behaviour directs disturbed inbound foragers back along their outbound path for a short distance before recovering and reorienting back to the nest. Supplementary Information The online version contains supplementary material available at 10.3758/s13420-023-00604-1.",
"conclusion": "Conclusions As highlighted by Cheng’s research (Cheng, 2022 ; Cheng et al., 2009 ; Freas & Cheng, 2022 ; Freas et al., 2019a , b , c ; Graham & Cheng, 2009 ; Islam et al., 2020 , 2021 , 2022 ; Schultheiss et al., 2016 ), research on ant species living in different ecologies has revealed a rich toolkit of navigational mechanisms that function together to produced impressive navigational behaviours. Here we show that Formica \n obscuripes attend to both the visual panorama and a path integrator for orientation, with the pheromone’s presence acting as a cross sensory, non-directional verification cue, confirming the correct path and continued orientation to these other strategies. Backtracking behaviour in this species is elicited by a combination of unfamiliar terrestrial cues and the absence of the pheromone, thus operating on a combination of mechanisms observed in both solitarily and socially foraging species. We also characterise a novel form of goalless orientation, an initial retreating behaviour that is modulated by the forager’s path integration system. The behaviour directs disturbed foragers back along their outbound column route for a short distance before recovery, presumably as a defensive response to threat.",
"introduction": "Introduction Extensive research by Ken Cheng and colleagues on navigation in ants has highlighted the value of a comparative ecological approach in which mechanisms are studied in the context of their function in the natural environment (e.g., Bühlmann et al., 2011 , 2020 ; Cheng et al., 2009 , 2012 , 2014 ; Cheng, 2022 , 2023 ; Freas & Cheng, 2022 ; Freas et al., 2018 , 2019a , b , c ; Schultheiss et al., 2016 ). Comparisons between ant species have revealed many similarities that exist across a wide range of environments and foraging ecologies, with the presence of a common underlying navigational toolkit of concurrently operating strategies (Bühlmann et al., 2011 ; Cheng et al., 2009 ; Freas & Spetch, 2023 ; Wehner, 2020 ). Of these, the most frequently observed across ant species consists of an updating vector maintained by the path integration system reliant on a celestial compass (Collett & Collett, 2000 ; Wehner et al., 1996 ; Wehner, 2020 ). When visual terrestrial cues are available, ants often also use these visual landmarks, by learning and retaining panoramic views at the nest and along foraging routes for later comparison when orienting (Cheng et al., 2009 ; Freas & Cheng, 2018 ; Freas & Spetch, 2019 ; Narendra et al., 2007 ; Schultheiss et al., 2016 ; Wystrach et al., 2011 ; Zeil, 2012 ; Zeil & Fleischmann, 2019 ). While these commonalities across species are interesting, just as intriguing are mechanistic differences expressed by various ant species to solve similar spatial challenges. Such differences can arise from the cue availability in the local environment but are also closely tied with the foraging ecology of each individual species (Bühlmann et al., 2011 ; Cheng et al., 2014 ; Freas at al., 2019a , b , c ; Schwarz & Cheng, 2010 ). Within ants, many well-studied species forage solitarily and must navigate alone, yet trail following ants rely upon many of the same navigational mechanisms with the additional complexity of the pheromone trail also helping to direct movement. Much of what we know regarding the strategies of navigating ants is based on solitarily foraging species that rely heavily on the visual cues of the celestial compass and the surrounding panorama (the 360º scene given ants see in a ~300º field of view) to navigate (Cheng et al., 2009 ; Freas et al., 2021 ; Narendra et al., 2017 ; Warrant & Dacke, 2016 ; Wehner, 2020 ; Wystrach et al., 2013 ). However, there has been parallel interest focused on how trail following ants integrate both individual and communal types of information to navigate (Almeida et al., 2018 ; Aron et al., 1993 ; Czaczkes et al., 2019 , 2022 ; Freas & Spetch, 2021 ; Grüter et al., 2011 ; Jones et al., 2019 ; Minoura et al., 2016 ; Middleton et al., 2018 ). Recent navigational work in a Sonoran Desert harvester ant, Veromesser pergandei , presents a demonstration of the underlying mechanistic differences observed between socially and individually foraging species . V. \n pergandei foragers exhibit a hybrid foraging structure, termed a “column and fan” (Plowes et al., 2013 , 2014 ). These ants begin their foraging journey socially, along a pheromone-marked column before individuals exit the pheromone and fanning out several meters to forage alone. Foragers maintain a path-integration-derived vector both in the column and the fan (Freas et al., 2019a , b ). Once food is collected, foragers return first to the end of the column (column head) using a path-integration-derived local vector (Freas et al., 2020 ), then shift their headings to follow their global vector along the pheromone column to the nest (see also Flanagan et al., 2013 , for potential evidence the phenomena may also be present in trunk trail systems). This pheromone mechanism mediating inhibition of part of the path integration system is in stark contrast to local vectors in solitarily foraging species, which are instead mediated by familiar views along the route (Collett et al., 1998 ; Webb, 2019 ). Local vectors represents but one of multiple mechanistic differences underlying similar path integration-linked behaviours, including maintaining orientation, backtracking and partial vector suppression, all of which are mediated in V. \n pergandei by the presence of the pheromone rather than panorama views as is the case for solitary foraging ants (Collett et al., 1998 ; Freas et al., 2019a , b , c , 2020 ; Freas & Spetch, 2021 ; Plowes et al., 2019 ; Wystrach et al., 2013 ). In fact, with regard to view-based navigation, V. \n pergandei shows no evidence of using view alignment of the panorama to orient, despite living in a visually cluttered environment where sympatric solitarily foraging ant species actively rely on these same views to home (Freas et al., 2019a , b , c , 2021 ). This hybrid foraging structure, with individuals relying on a pheromone column before leaving the column to navigate solitarily during distinct periods of the journey, is not exclusive to V. \n pergandei . The western thatching ant ( Formica \n obscuripes ) also initially relies on a pheromone-marked column when leaving the nest, which shares some similarities to the column-and-fan structure of V. \n pergandei . Yet in F. \n obscuripes , the pheromone column extends out to stands of Artemisia bushes where foragers either climb into these bushes to farm Sternorrhyncha (collecting honeydew from aphids) or fan out along the surrounding ground and branches to hunt for other arthropods (Fig. 1 ). F. \n obscuripes is known to inhabit a variety of visually cluttered environments across its range (Glasier et al., 2014 ; Mackay & Mackay, 2002 ; Wheeler & Wheeler, 1986 ), suggesting it may rely on panoramic views to orient. Yet, little is known of the navigational abilities of F. obscuripes , and the navigational mechanisms they employ while travelling in either the presence or the absence of their pheromone. Fig. 1 Photos of the F. \n obscuripes column with distinct segments from the nest to a group of bushes where foragers spread out to collect food at 24 m. This non-straight-line route contains a 40° counter-clockwise turn at 7 m from the nest, as well as a 7° clockwise turn at 16 m along the column. Photos exhibit the degree of visual cues available to navigating individuals and the changes in clutter over the route Our initial aim in the current study was to characterize the navigational capabilities of F. \n obscuripes , a species inhabiting an array of habitats across a large geographic range throughout North America, yet has not been studied for its navigational behaviour (Wheeler & Wheeler, 1986 ). Specifically, we sought to discover if the mechanisms underlying navigation in the presence or absence of the pheromone cue, including orienting, path integration and backtracking, aligned with the only other studied fan-and-column foraging species, Veromessor pergandei . We began by conducting distant displacement tests to determine if heading behaviour was consistent with celestial compass-based path integration. Observations during these tests uncovered an interesting and previously undocumented behaviour of inbound foragers where foragers initially ‘retreat’ after release by first travelling in their outbound direction before altering their headings to celestial-compass-based inbound orientation, consistent with following a vector. We have classified this behaviour as retreating as it consists of abandoning active navigation to the current goal (nest). Retreating is often classified in outbound ants when foragers stop homing to a resource to return to the nest when cues change (i.e., when the celestial compass is altered; Freas et al., 2017b ), however, the consistency between outbound and inbound retreating is the abandonment of the current goal and a return in the direction recently travelled. Typically, when outbound orientation is observed in inbound ants, it is associated with backtracking, a behaviour that occurs when foragers are close to the nest and have a near-zero path integrator state (Freas et al., 2019a ; Plowes et al., 2019 ; Wystrach et al., 2013 ). Yet here the observed outbound headings occurred in foragers collected all along the column when much of their accumulated vector remained. Additionally, backtracking is considered a form of directed search, which persists as the forager’s search expands, inconsistent with this retreat behaviour (Müller & Wehner, 1994 ; Schultheiss et al., 2016 ; Wystrach et al., 2013 ). Here, outbound orientation was only temporary when foragers had most of their vector remaining, suggesting this behaviour is distinct from backtracking. We chose to characterize retreating behaviour, and the conditions under which it occurs, in a separate group of tests in addition to establishing the general navigational capabilities of this species.",
"discussion": "Discussion The direction of initial orientation when released at distant sites depended upon the location along the column at which the inbound ants were collected. For inbound ants collected within the column, distant testing uncovered an ‘initial retreat’ behaviour, in which individuals released distantly either on the reference circle board or on the ground initially oriented in the outbound compass direction. Interestingly, this retreating behaviour did not occur when collected near the nest (0 m) or at the column head (24 m). In both of those cases, initial orientation was random. Analysis of forager paths over longer distances revealed that individuals in all groups recovered from their initial retreat or search behaviour and showed significant orientation by 3 m. However, the direction of orientation again depended on collection location. For ants collected within 11 m of the nest, orientation was in the outbound direction, reflecting backtracking behaviour. However, ants collected at 14, 16 or 24 m from the nest (with over 50% of their column distance remaining) oriented in the inbound direction. This suggests that backtracking in this species relies, at least in part, upon the remaining portion of their accumulated global vector.\n\nDiscussion Our retreating results suggest the behaviour contains four predictable aspects. (1) Retreating occurred after collection from within the column but not the nest or column head. (2) Retreating is distinct from backtracking behaviour. (3) Retreating is not elicited via navigational uncertainty. (4) Retreat was in the direction of current route segment and not a global vector direction. Combined, the results point to retreat as being a goalless directed orientation behaviour elicited by aversive events and informed by the forager’s current vector state and guided by the celestial compass. Furthermore, F. \n obscuripes foragers show evidence both of vector maintenance and panorama use while navigating both on and off the pheromone cue. Importantly, the pheromone’s presence appears critical to continued orientation to these other cues with laterally displaced foragers ultimately abandoning inbound homing when not on the pheromone trail.\n\nDiscussion Backtracking behaviour in ants collected just before entering the nest and tested distantly was substantially eliminated when the pheromone cue was present (compare Figs. 2 b, 3 , 4 , 5 and 6 c), and instead the ants oriented in the inbound direction. The elimination of backtracking did not depend on the directionality of the pheromone trail because rotation of the board containing the pheromone trail had no effect. These results (along with the findings from Experiment 1 ) indicate that F. \n obscuripes exhibits backtracking based on a combination of the factors previously observed in other ant species. Backtracking was observed when at an unfamiliar location with less than 50% homeward route remaining, indicating that recent exposure to the nest’s panorama was not a critical trigger as reported in M. \n bagoti (Wystrach et al., 2013 ; OSM Fig. 1 ). However, the results of testing on the board above the column, together with the distant pheromone testing results, suggest that F. \n obscuripes can rely either on the familiarity of the current panorama or the presence of the pheromone when deciding to not backtrack. Backtracking occurs only in the absence of both cues, which is distinct from both solitarily foraging ants and from trail following V. \n pergandei (Wystrach et al., 2013 ; Freas et al., 2019a ).\n\nGeneral discussion Summary Our findings can be separated into two themes. First are those concerning the general navigational abilities of Formica \n obscuripes during foraging and how it compares to other ant species. Second is a discussion of the novel directed retreating behaviour and its underlying mechanisms as well as how it fits within this species’ foraging ecology. On the navigation theme, distant displacement headings suggest that the ants maintain a path-integration-derived vector while inbound on the pheromone column, whereas displacements on, above and near the column show a reliance on familiar panoramas for guidance. Additionally, there is clear evidence of backtracking behaviour in this species distinct from their initial retreating behaviour. Backtracking appears to be mediated by a combination of visual cues and the pheromone, ultimately showing a combination of mechanisms observed in other ant species where this behaviour has been characterised. Navigational strategies Path integration The maintenance of a path-integration-derived vector, reliant on the celestial compass, is well modelled in ants (Collett & Collett, 2000 ; Wehner, 2020 ; Wehner et al., 1996 ). Additionally, the presence of a path integrator is known to run both in many individually foraging species as well as in socially foraging species while on a pheromone trail (Collett & Collett, 2000 ; Freas et al., 2019b ; Heinze et al., 2018 ; Wehner & Srinivasan, 2003 ). In socially foraging species, this vector is thought to provide directional information on the trail as non-bifurcating pheromone trails lack inherent polarity (Czaczkes et al., 2015 ; Freas et al., 2021 ; Minoura et al., 2016 ). Much like other ant species, F. \n obscuripes foragers show evidence of vector maintenance, using both directional compass cues as well as distance estimates to orient when placed at a distant unfamiliar site, both aspects of a continuously operating path integrator. Forager paths showed clear orientation to the compass direction associated with their inbound route (after their initial retreat) when they had > 50% of their column distance remaining. In contrast, once foragers had completed 50% of their inbound column distance, they began to orient in the opposite compass direction, consistent with backtracking behaviour, which is known to rely on the celestial compass and odometer cues (Freas et al., 2019a ; Plowes et al., 2019 ; Wystrach et al., 2013 ). Additionally, when inbound foragers are collected at the nest, with their path integrator directionally uninformative, and displaced distantly to a pheromone-marked board, they continued to orient to their inbound vector direction rather than backtrack. This inbound direction was observed even when the pheromone board was rotated by 180°, thereby ruling out control of the inbound behaviour by directional cues from the pheromone trail. These results indicate two aspects of the relationship between the pheromone cue and the forager’s path integrator, which are identical to interactions observed in V. \n pergandei (Freas et al., 2021 ). First, it is further evidence of a lack of inherent pheromone directionality as there was no difference in headings with the pheromone in agreement or conflict with the vector compass direction with the lack of inherent polarity in straight pheromone trails being established in multiple ant species (Czaczkes et al., 2015 ; Freas et al., 2021 ; Minoura et al., 2016 ). Instead, the presence or absence of the pheromone, along with the proportion of the vector completed, dictates whether foragers continue to orient to their homeward vector or begin backtracking. Secondly, like V. \n pergandei (Freas et al., 2021 ) , foragers continued to orient to the inbound vector compass direction despite having a directionally uninformative vector state. Given the inherent accumulation of error in the path integration system, when the remaining vector state is near zero, yet the forager is still in contact with pheromone, it is likely advantageous to continue orienting in the inbound vector direction, rather than engage in search or backtrack as the pheromone’s presence indicates the nest has not yet been reached. However, the memory dynamics, such as whether foragers rely on a reloading of a long-term memory during this testing, remain unknown. The results are ultimately indicative that the pheromone acts as a context cue in how foragers choose their headings based on their path integrator, just as observed in V. \n pergandei (Freas et al., 2021 ). Panorama cues Forager headings in multiple local displacement tests show evidence of the use of familiar panoramic views to orient while near known locations. In conditions where foragers were released back into the column at 14 m, individuals oriented in the direction of the route’s views even when it conflicted with their remaining vector state (7-m condition), suggesting this vector is being overridden by view alignment. When we collected the full paths of foragers released back into the pheromone column from 14 m, foragers correctly follow this column even in the absence of a corresponding vector cues. As the path integrator is continuously running, foragers collected at the nest (0 m) were likely accumulating a directionally conflicting vector in opposition with these views, yet still showed little hesitation to follow the inbound column. Just as with the path integration system, the presence of the pheromone appears to act as a context cue in relation to view alignment strategies. Though importantly, these on-pheromone paths could be accomplished through initially recognising the polarity of the pheromone trail via views or a vector (depending on condition) and then using the pheromone alone to navigate. In the above-column and 2-m lateral displacements, which were both off the pheromone, headings were initially oriented to the correct inbound view direction (up to 1 m). However, 2-m lateral displacement paths suggest that foragers ultimately could not follow these views home or to the column in the absence of the pheromone and instead they appeared to quickly abandon this orientation in favour of backtracking. Results indicate that, like other trail following ants (Czaczkes et al., 2015 ; Freas et al., 2021 ; Minoura et al., 2016 ), in F. \n obscuripes the pheromone cue alone contains no directional information, yet its presence informs how foragers use their directionally based navigational systems. Specifically, the pheromone’s presence acts to decrease uncertainty, consequently increasing the weighting given to navigational systems (path integration or view memory) over search behaviours, including backtracking. In the absence of the pheromone cue, weighting of these same navigational systems is suppressed, leading foragers to engage in search despite familiar views or a remaining path integrator. In this way, the pheromone acts as a verification or reassurance cue confirming with its presence that the foragers are travelling in the correct direction and should continue to follow these cues. The pheromone’s function to reassure foragers they are on the correct path has been documented in other ant species (Czaczkes et al., 2011 ; Freas et al., 2021 ; Wetterer et al., 1992 ) suggesting it may play a similar function in all trail following ants. Backtracking Navigational strategies (path integration and view memories) typically fail to return individuals to the exact location of the nest. Thus, navigating ants employ a number of back-up mechanisms during this final stage of their journey to pinpoint their goal. These include systematic search behaviour (Schultheiss & Cheng, 2011 ; Schultheiss et al., 2015 ; Wehner & Srinivasan, 1981 ), which occurs both in familiar and unfamiliar locations, and a behaviour called backtracking. Backtracking is thought to be widespread in navigating Hymenoptera (Collett & Collett, 2009 ; Collett et al., 1993 ; Freas et al., 2019a ), yet its mechanisms have only been described in two ant species, a single solitarily foraging species, Melophorus bagoti (Wystrach et al., 2013 ) and more recently a single fan-and-column foraging species, V. \n pergandei (Freas et al., 2019a ; Plowes et al., 2019 ). This behaviour sees inbound foragers, displaced just before entering the nest to an unfamiliar location, orienting in the opposite (i.e., outbound) direction they were previously heading before displacement, instead of a random direction indicating systematic search. Backtracking behaviours are theorised to aid foragers who have overshot the nest and are now in an unfamiliar area, leading them to return back along their route to reach more familiar locations. This behaviour only occurred in M. \n bagoti foragers under the combination of three criteria: (1) individuals had a path integrator state near zero, (2) individuals were presented an unfamiliar terrestrial panorama, and (3) individuals had recently been exposed to the nest panorama. These triggering conditions are not ubiquitous across ant species, with V. \n pergandei using the presence/absence of the trail pheromone instead of panorama familiarity to trigger backtracking. Additionally, V. \n pergandei did not require a path integrator state at or near zero, using instead the proportion of the homeward route (~75%) they ran off, which triggers backtracking, meaning the nest panorama is not a universal trigger (Freas et al., 2019a ). Here, F . obscuripes show a combination of the criteria observed in other ant species. First foragers were observed to backtrack when displaced to a distant, unfamiliar location when they had completed between 54% and 100% of their homeward route, with foragers collected even 11 m from the nest exhibiting backtracking. This is almost double the remaining path integrator proportion (46% vs. 25%) observed in V. \n pergandei and well beyond the nest’s panorama critical for backtracking in M. \n bagoti (OSM Fig. 1 ) . Second, foragers appear to use familiar panorama views when deciding to backtrack as they do not exhibit backtracking when exposed to the familiar panorama just above the column with no accompanying pheromone cues. Foragers can also use the presence of the pheromone when deciding to backtrack as zero vector foragers displaced distantly to an unfamiliar panorama onto the pheromone do not backtrack and instead orient to their inbound vector. This suggests that backtracking in F . obscuripes meets backtracking criteria of both previous species. Like V. \n pergandei, they clearly use the proportion of their column they have run off and not the nest panorama to initiate backtracking. Yet unlike V. pergandei, in this species backtracking only occurs in the absence of both the pheromone and a familiar panorama. One complication of this description of backtracking is our results during 2-m lateral displacements. Here, foragers with little or no column distance remaining were presented a panorama that only differed slightly from their route panorama at 14 m (OSM Fig. 1 ), with foragers showing an ability to orient to these inbound views for at least 1 m. Yet by the time these foragers exited the grid, both groups of foragers (7 m and 0 m) were oriented in outbound directions consistent with backtracking. As noted above, this suggests that the presence of the pheromone is likely necessary as a verifier for continued view-based homing. In the pheromone’s absence, foragers will still ultimately choose to backtrack to attempt re-enter the pheromone column rather than face the challenge of travelling to the nest off the pheromone trail. Retreating behaviour Formica obscuripes appears to be more sensitive to disturbance than many other species which typically don't show retreating in response to either collection or testing on an unfamiliar substrate. The observed initial retreating behaviour in F . obscuripes exhibited four predictable characteristics. First, retreating occurred after collection at all points within the column length but not at its two ends (at 0 m and 24 m), where forager headings were initially random. Second, this retreat was distinct from backtracking behaviour. Third, foragers exhibited retreating behaviour both when the available navigational cues upon release were familiar as well as when individuals were released distantly to an unknown location, suggesting the behaviour is not elicited via navigational uncertainty. Finally, while directional differences between the global vector and the current route segment were often small, retreating foragers in multiple conditions only significantly oriented to their current route segment when retreating and not a global vector direction. Together the findings point to a celestial compass based, non-goal-directed orientation response elicited by the experimental manipulations and informed by the forager’s current vector state. This suggests retreating behaviour is a response to aversive events meant to facilitate escape while remaining on the pheromone marked column. This would align with retreating only being observed when their vector state indicates they were positioned within the column and not its ends. If a forager experiences an aversive event with a vector state indicating it is at the column head, retreating in an outbound direction where no pheromone exists would push them to travel off the column, increasing the chance they become lost. This may also explain the lack of retreating with a near zero vector state (while these same foragers still exhibited backtracking). With the safety of the nest so close, retreating away from the nest may also be disadvantageous for an anti-predatory response, leading to longer periods outside of the nest. This is in clear contrast to backtracking behaviour where the absence of the pheromone with a near-zero vector state means the forager may have passed the nest and should search the in opposite compass direction. An important aspect of the observed retreating behaviour is that it occurs regardless of the level of navigational uncertainty present when orienting. Retreating occurs at the distant testing site or when displaced back into their foraging route, suggesting the degree of familiar cues present (view memories and pheromone presence) do not influence this behaviour. Additionally, displacements in route-following ants to other parts of their foraging route, thus placing the expect views out of order, have been shown to influence navigational uncertainty and increase hesitation behaviours (Schwarz et al., 2020 ). Yet here we observed no difference in retreating behaviour based on whether foragers were released to a familiar but unexpected view sequence of the inbound route (7-m or 14-m foragers released back on column at 14 m), suggesting that this uncertainty also had little or no influence on retreating. Instead, retreating appears to be triggered by the collection-and-release procedure, with foragers released directly onto the ground or board exhibiting the behaviour. Once we implemented the procedure to force foragers to descend 10 cm before choosing a heading, delaying the period between release and foragers choosing a heading, we observed a significant decrease in initial retreating behaviour. Additionally, while navigational cue presence was shown not to influence this behaviour, the familiarity of the ground’s substrate did appear to affect retreating. When we released foragers just above their foraging column on a wooden board, they retreated even in the presence of familiar panorama cues. Yet, we were able to extinguish this retreating behaviour by spreading a familiar soil substrate (from a location off the pheromone column) over the board’s surface. These results suggest that the unfamiliar wooden substrate caused a neophobic response from the ants, with continuous contact with the substrate being aversive and eliciting retreat, similar to the aversive effects of the original release procedure. Finally, initial outbound orientation at distances between 4.5 m and 16 m on the column would represent points at which the forager is not close to the nest and that when responding to an aversive incident, they can retreat along the column while remaining on the pheromone. This means foragers should orient, not in the outbound of their full vector but to the outbound direction of their current route segment along the column, in order to remain on the pheromone. We see evidence of this orientation to the current route in two conditions: the distant initial headings at 4.5 m and 14 m, where foragers are only oriented to the current outbound route compass direction and not the full outbound vector. In many of the conditions where we observed this retreat, the directional differences between the global outbound vector and the outbound direction of the current route were quite small, thus making differentiating between these two directions difficult using 95% CIs; yet we find multiple instances of orientation only to the current route alone while in no condition do we find orientation to the outbound global vector alone. Coupled with the other instances of inbound route-segment orientation such as in the 3-m paths of distantly displaced column head (24 m) ants, these findings indicate that a mechanism may exists for these foragers to orient in relation to part of their vector and not their full vector state, similar to foragers orienting to their final path segment during backtracking (Freas et al., 2019a ; Wystrach et al., 2013 ) or when orienting via only ocelli (Schwarz et al., 2011 ). Orientation to vector segments rather than the global vector, in particular during the inbound route, would allow foragers to retrace the non-straight pheromone trail instead of leaving the pheromone to travel in a straight-line to the nest as a shortcut. How this segment-based orientation interacts with the path integration system is interesting and merits further research."
} | 8,408 |
22970303 | PMC3435296 | pmc | 4,227 | {
"abstract": "To improve their nutrition, most plants associate with soil microorganisms, particularly fungi, to form mycorrhizae. A few lineages, including actinorhizal plants and legumes are also able to interact with nitrogen-fixing bacteria hosted intracellularly inside root nodules. Fossil and molecular data suggest that the molecular mechanisms involved in these root nodule symbioses (RNS) have been partially recycled from more ancient and widespread arbuscular mycorrhizal (AM) symbiosis. We used a comparative transcriptomics approach to identify genes involved in establishing these 3 endosymbioses and their functioning. We analysed global changes in gene expression in AM in the actinorhizal tree C. glauca . A comparison with genes induced in AM in Medicago truncatula and Oryza sativa revealed a common set of genes induced in AM. A comparison with genes induced in nitrogen-fixing nodules of C. glauca and M. truncatula also made it possible to define a common set of genes induced in these three endosymbioses. The existence of this core set of genes is in accordance with the proposed recycling of ancient AM genes for new functions related to nodulation in legumes and actinorhizal plants.",
"introduction": "Introduction Mutualistic interactions between plants and microorganisms are an essential and widespread adaptive response whose origin can be traced back to land colonisation by plants: fossil evidence demonstrates that ∼450 million years ago primitive plants were already associated with fungi to form arbuscular mycorrhizal (AM) symbioses [1] . Today, more than 80% of terrestrial plants form AM in association with Glomeromycota fungi. AM fungi colonise the root cortex and differentiate intracellular structures inside cortical cells – arbuscules or coiled hyphae – which play a crucial role in nutrient exchange. AM significantly improve plant mineral nutrition, increasing growth and tolerance to environmental stresses including pathogens [2] . More recently, ∼60 MY ago, certain plants evolved the ability to form endosymbiotic associations with nitrogen-fixing bacteria to improve their nitrogen acquisition. The most intricate of these symbioses leads to the formation of a new organ, the root nodule, where bacteria hosted in a favourable environment inside plant cells are able to fix enough atmospheric nitrogen to sustain plant growth without any other nitrogen source. The ability to form root nodule symbioses (RNS) evolved only in fabids and gave rise to two main types of symbioses: (1) rhizobial RNS involve gram negative proteobacteria collectively called rhizobia that associate with plants from the Fabaceae superfamily and a few species from the genus Parasponia ( Cannabaceae ), (2) actinorhizal symbioses combine fabids distributed into 8 families, collectively called actinorhizal plants, and the gram positive actinomycete Frankia \n [3] – [5] . Nodulation emerged several times independently within the Fabidae suggesting that the common ancestor of this clade acquired a still-unknown predisposition towards RNS [4] . Most genes involved in nodulation are similar to genes involved in other processes, suggesting that RNS evolved by recycling a variety of pre-existing genetic mechanisms. Genes controlling the development of rhizobial infection threads are probably derived from genes controlling pollen tube growth [6] . Many genetic mechanisms making it possible to accommodate symbiotic bacteria originate in more ancestral AM symbiosis [4] , [7] , [8] : the symbiotic signals emitted by rhizobia and AM fungi are chemically related [9] . In addition, part of the signalling pathway responsible for signal transduction in host plants in response to recognition of the microbial partner is shared between AM, rhizobial and actinorhizal symbioses [8] , [10] – [13] . We used comparative transcriptomics to identify genes induced during AM and nodulation (actinorhizal or rhizobial) in several plants, including legumes [14] , [15] , rice [16] and the actinorhizal tree Casuarina glauca \n [17] . As no data on AM in actinorhizal plants were available, we characterised the establishment of AM between the actinorhizal tree Casuarina glauca and Glomus intraradices and analysed its transcriptome profile. By comparing genes induced in AM in Medicago truncatula, rice and C. glauca we identified a group of genes induced in AM in these three distant species and a group of genes induced during AM, rhizobial and actinorhizal nodulation. Those genes were clustered in functional groups that may play crucial roles in the establishment and the functioning of the three endosymbioses and how they work.",
"discussion": "Results and Discussion Establishment of AM symbiosis between C.glauca and G. intraradices \n First we characterised the colonisation kinetics of C. glauca by the AM fungus G. intraradices . Three-week old plants were transferred to pots containing soil inoculated with G. intraradices . Five plants were analysed for their mycorrhizal status every 3 days from two weeks after inoculation to 48 days after inoculation (dai). We observed a regular increase in the percentage of plants showing intraradical fungal structures over time; all plants were colonised from 44 dai ( Figure 1A–B ). The type of fungal structures observed on the plant root varied over time. Up to 21 dai only intraradical hyphae were observed. At 23 dai, coiled hyphae, arbuscules and vesicles appeared ( Figure 1C-E ). From these observations, plants 45 dai were selected to characterise the C. glauca transcriptome response to AM symbiosis. 10.1371/journal.pone.0044742.g001 Figure 1 Analysis of AM establishment in C. glauca. (A) Percentage of plants showing internal AM structures; (B) Average mycorrhization rate in plants showing internal AM structures (bars: standard deviation); (C–E) Analysis of intraradical structures in roots of C. glauca roots after inoculation with G. intraradices: (C): quantitative analysis (D–F) CLSM images acquired on roots 45 days after inoculation showing extensive fungal colonisation, the presence of arbuscules (D), coiled hyphae (E) and vesicles (F). Bar = 20 mm. Gene expression in C. glauca AM and comparison with other AM symbioses In order to identify the C. glauca genes regulated by AM symbiosis, a 15 K C. glauca genechip [17] was hybridised with cDNA from control (non inoculated) and roots inoculated with G. intraradices . 124 genes were down-regulated and 430 up-regulated in C. glauca AM roots (FC≥2, p-value≤0.01). Microarray data were confirmed by Q-PCR on genes showing various expression levels ( Table S1 ). We were particularly interested in identifying genes involved in the intracellular accommodation of symbionts. While down-regulation of some gene might be important for intracellular accommodation of symbionts (for instance defence-related genes), we focused our analysis on genes that were induced. Of these, 324 appeared to be from C. glauca and 106 from G. intraradices ( Tables S2 and S3 ). Homologues of known specific AM marker genes PT4 (Phosphate transporter 4), BCP1 (Blue Copper Protein 1), or SCP1 (Serine CarboxyPeptidase 1) [15] , [18] were induced in our dataset, thus validating the experiment ( Table S2 ). CGCL918Contig1, a presumed homologue of the aquaporin NIP1 (Nodulin 26-like intrinsic protein 1) specifically expressed at a low level in the arbuscule-containing cells [18] was also induced in our data. This might suggest that our experimental set made it possible to detect genes with low levels of expression. We then compared genes up-regulated in AM in C. glauca , the model legume M. truncatula \n [15] and the monocot O. sativa \n [16] . This analysis revealed 84 C. glauca genes up-regulated in AM similar to M. truncatula and O. sativa AM-induced genes ( Figure 2A–C , Table S4 ). These may represent some core functions needed for AM symbiosis. 10.1371/journal.pone.0044742.g002 Figure 2 Transcriptional regulations in M. trucatula , O. sativa and C. glauca AM. (A) Number of genes up-regulated in AM in these different species; (B) Functional distribution of the 84 AM-induced genes in C. glauca and conserved in M. truncatula and O. sativa ; (C) Induction of AM markers in C. glauca 48 days after inoculation by G. intraradices. \n The cluster most represented corresponded to proteases (27 C. glauca unigenes), in accordance with the important role played by protein turnover in AM [16] , [19] – [21] . Among the conserved proteases, we found subtilisin proteases of the S08A family. None of these correspond to Cg12 the C. glauca subtilase that is specifically expressed during plant cell infection by Frankia \n [22] , [23] . In Lotus japonicus , two members of this family, LjSbtM1 and LjSbtM3, are found in the peri-fungal space and are involved in AM development [24] . Proteases may be responsible for selective processing of substrates present in the peri-fungal space, generating peptides recognised by leucine-rich repeat receptors involved in the AM interaction such as PaNFP or SYMRK. Interestingly, a gene encoding a putative receptor with LRR repeats (CG-R02f_036_O05) was induced in AM in all three plants. Proteases may also be involved in the cell wall loosening and cell remodelling associated with mycorrhizal infection [24] , or in arbuscule senescence [25] . Ten C. glauca sequences corresponding to carboxypeptidases belonging to the papain C1A family [26] may belong to this category as this family contains senescence-associated proteins such as AtSAG12 and MtCP1-6 [25] . Seven genes encoding putative members of the cytochrome P450 family were among the conserved genes. Most of them belong to the CYP71 family that is usually associated with triterpenoid biosynthesis. Triterpenoids play diverse biological roles, including antifungal and antibacterial (Fukushima et \n al ., 2011). Two other singletons annotated “ent-kaurene oxidase” belong to the P450 class and were conserved. Interestingly, a comparison with the Arabidopsis proteome database revealed homologies with ENT-KO, a member of the CYP701A subfamily involved in the gibberellin biosynthetic pathway (Sawada et \n al ., 2008; Achard & Genschik, 2009; X.-H. Gao et \n al ., 2011). Moreover, another gene (corresponding to CG-R02f_045_J11, Mtr.31291.1.S1_at, OS07G39270) annotated as GeranylGeranyl Pyrophosphate Synthase, is homologous to AtGGPS1 , which is also involved in Gibberellin biosynthesis (Okada et \n al ., 2000). These results are consistent with the Gibberellin biosynthesis regulation occurring in AM (Güimil et \n al ., 2005; Gomez et \n al ., 2009; Schäfer et \n al ., 2009; Fiorilli et \n al ., 2009; Hogekamp et \n al ., 2011) and with the postulated role of this phytohormone as a compatibility factor in AM [27] . Another important group that was conserved were transporters: eight C. glauca genes belong to this category. Among them is the aforementioned MtPT4 (Mtr.43062.1.S1_at; [28] and its orthologues in rice (OsPT11; OS04G10800; [29] ) and C. glauca (CgPT4, CG-GI1f_006_M09)) encoding a high affinity phosphate transporter specifically expressed in the peri-arbuscular membrane and responsible for the symbiotic transport of phosphate in M. truncatula \n [30] . Another transporter (CGCL918Contig1) shared 73% identity with MtNIP1, an aquaporin specifically expressed in cells containing arbuscules [31] , which has been suggested as being involved in inorganic N uptake into plant cytoplasm [31] , [32] . Other genes related to transport encode putative oligopeptide transporters potentially involved in the intake of small peptides produced by the degradation of fungal proteins during the senescence of arbuscules, or in the intake of signal peptides [18] . Two putative members of the ABC-transporter family are also among the conserved genes. CG-N02f_013_P06 shares 80% homology with MtSTR2 (for stunted arbuscules; Zhang et \n al ., 2010). MtSTR2 interacts with MtSTR to form a functional heterodimeric transporter that co-localises at the peri-arbuscular membrane and is essential for arbuscule development [33] . CGCL1417Contig1 presents 62% identity with AtPGP1 (P-GlycoProtein 1), 63% with AtPGP4 and 63% with AtPGP16. These members of the P-GLYCOPROTEIN (PGP) transporters family are able to transport a wide range of molecules [34] . Another conserved gene cluster corresponds to chitinases (4 C. glauca unigenes). Interestingly, CGCL506Contig1 presents 84% similarity with MtCHITIII-3, which is specifically expressed in cells containing arbuscules in M. truncatula \n [35] . Disruption of its expression resulted in a higher root colonisation by G. intraradices \n [36] . This chitinase may be involved in the modulation of chitin elicitors, and have an impact on signalling between the plant and fungus. Genes involved in lipid metabolism are also conserved between the three species; this is in accordance with the important role played by lipid metabolism during synthesis of the peri-arbuscular membrane concomitant to internalisation of the fungi, as well as in recycling lipids from degenerating arbuscules [2] . A purple acid phosphatase, CG-GI1f_003_A02, sharing 86% identity with AtPAP10 and 88% identity with MtPAP1, was also identified. These proteins are involved in phosphate nutrition probably through phytate degradation [37] , [38] . Within conserved elements, we identified several signalling components such as protein kinases, a U-box protein from the same family as LIN, a protein involved in nodulation [39] , and transcription factors from the GRAS and AP2/ERF family. In conclusion, our study highlights key biological processes that were conserved throughout plant evolution, and that are probably essential for AM establishment and functioning. Comparison of gene expression in AM, rhizobial and actinorhizal symbioses In order to analyse the potential conservation of the molecular mechanisms involved in AM and actinorhizal symbioses, we compared genes induced in these two symbioses in C. glauca . A simple spreadsheet application named Casuarina Transcriptome Compendium (CTC; Table S5 ) was created for comparative transcriptomics in C. glauca (for guidelines, see File S1 ). CTC allowed us to identify 94 genes up-regulated both in AM and actinorhizal nodules (FC> = 2, p-value< = 0.01, Figure 3A ; Table S6 ). Functional classes recovered were partially similar to those found when comparing AM and rhizobal symbioses in Legumes [19] , [40] . RT-qPCR was used to confirm the induction of a subset of these genes in both interactions ( Table S6 ). 10.1371/journal.pone.0044742.g003 Figure 3 Conservation of gene expression in AM and root-nodule symbioses. (A) Transcriptomic comparison between C. glauca genes up-regulated in AM and actinorhizal nodules; (B) Conservation of genes up-regulated in both AM and nodules in C. glauca and M. truncatula ; (C) Functional classification of the 24 conserved genes up-regulated during AM, actinorhizal and legume-rhizobium symbioses. In order to compare the set of genes involved in AM and root nodule symbioses in both legumes and actinorhizal plants, we compared the genes up-regulated in AM and actinorhizal symbioses in C. glauca to those up-regulated in both nodules and AM recently identified in the model legume M. truncatula (respectively 51 K and 61 K Affymetrix geneChip) [14] , [15] . Twenty-four C. glauca genes induced in AM roots and nodules (MycUp/NodUp) presented significant sequence homology with M. truncatula MycUp/NodUp genes ( Figure 3B–C ; Table S7 ). These genes might represent part of the heart of endosymbioses, conserved together in the ancestral AM symbiosis, legume-rhizobial and actinorhizal symbioses. Once again, genes encoding proteases formed the largest cluster (10/24), suggesting that proteases play a significant common role in the three endosymbioses. Interestingly, mutant screenings performed on model legumes did not yield any gene encoding protease involved in rhizobial or AM symbioses, either because mutants in these genes are lethal or because a high redundancy level is present. Maintenance of functional redundancy may reflect a need for very high expression levels for these genes in the context of endosymbioses [41] . Gene encoding transporters represented the second largest group. This group included the C. glauca STR2 homologue represented by 2 probes (CG-N02f_013_P06 and CG-GI1f_001_E14) corresponding to the same unigen. Zhang et \n al . (2010) did not report any phenotype during nodulation in the mtstr2 mutant. Our finding that this gene was among the core endosymbiotic gene set suggests that it may still play a subtler role in nodulation. Genes encoding peptide transporters and PGP family transporters were also up-regulated during all 3 endosymbioses. In conclusion, our work revealed genes that are induced in all three major plant endosymbioses: the ancient AM symbiosis, and the more recent RNS. This list represents genes probably linked to processes such as nutrient exchange, infection, and intracellular accommodation of the microsymbiont, and reflects the molecular tinkering that took place during evolution of nodulation using parts of ancestral AM mechanisms. Recycling signal transduction elements from AM to form RNS has previously been reported [7] , [12] , [17] . The corresponding genes were not recovered in our work as they are often not transcriptionally regulated ( Table S8 ). The genes we identified were strongly up-regulated in all endosymbioses and probably correspond to the end targets of the endosymbiotic programme. Further functional characterisation of these genes is needed to understand their precise role in the three different endosymbioses and to explain how they were recruited during the evolution of RNS."
} | 4,469 |
36578475 | PMC9791597 | pmc | 4,229 | {
"abstract": "Fatty acid-derived products such as alkanes, fatty aldehydes, and fatty alcohols have many applications in the chemical industry. These products are predominately produced from fossil resources, but their production processes are often not environmentally friendly. While microbes like Escherichia coli have been engineered to convert fatty acids to corresponding products, the design and optimization of metabolic pathways in cells for high productivity is challenging due to low mass transfer, heavy metabolic burden, and intermediate/product toxicity. Here, we describe an E. coli -based cell-free protein synthesis (CFPS) platform for in vitro conversion of long-chain fatty acids to value-added chemicals with product selectivity, which can also avoid the above issues when using microbial production systems. We achieve the selective biotransformation by cell-free expression of different enzymes and the use of different conditions (e.g., light and heating) to drive the biocatalysis toward different final products. Specifically, in response to blue light, cell-free expressed fatty acid photodecarboxylase (CvFAP, a photoenzyme) was able to convert fatty acids to alkanes with approximately 90% conversion. When the expressed enzyme was switched to carboxylic acid reductase (CAR), fatty acids were reduced to corresponding fatty aldehydes, which, however, could be further reduced to fatty alcohols by endogenous reductases in the cell-free system. By using a thermostable CAR and a heating treatment, the endogenous reductases were deactivated and fatty aldehydes could be selectively accumulated (>97% in the product mixture) without over-reduction to alcohols. Overall, our cell-free platform provides a new strategy to convert fatty acids to valuable chemicals with notable properties of operation flexibility, reaction controllability, and product selectivity.",
"conclusion": "4 Conclusions In this work, we demonstrated the application of CFPS-based in vitro systems for selective conversion of long-chain (C 12 –C 18 ) fatty acids to corresponding alkanes, aldehydes, and alcohols, which can be often used as fuels and commodity chemicals. Due to the open nature of cell-free systems, the same substrate can be selectively converted to different target products through the rational expression of related enzymes. First, a photoenzyme CvFAP was successfully expressed in vitro that enabled the decarboxylation of fatty acids to form alkanes under blue light. In particular, the conversion of palmitic acid and stearic acid in the cell-free systems reached 88% and 95%, respectively. Second, three CAR enzymes were separately expressed in vitro for the reduction of fatty acids to form fatty aldehydes, which can be further reduced to corresponding alcohols by endogenous ADH/AHR enzymes derived from E. coli cell extracts. In this CAR-based biotransformation system, we were especially able to selectively produce aldehydes and/or alcohols in one pot by choosing suitable CAR enzymes. For example, using a thermostable AnCAR, the proportion of fatty aldehydes such as hexadecanal and octadecanal could be accumulated more than 97% in the product mixture by heating deactivation of ADHs/AHRs. Since the activity of cell-free expressed CvFAP and CARs has been demonstrated in vitro , we expect that CFPS will be a promising and complementary approach for rapid expression and screening of improved enzymes from the engineered enzyme libraries, which were only carried out using in vivo expression systems as reported previously ( Kramer et al., 2020 ; Li et al., 2021 ). In summary, our cell-free platform has several key features. First, cell-free reaction is fast. It requires only hours to obtain target products, whereas a few days or weeks might be needed to grow cells for product formation in vivo . Second, the use of cell-free systems allows for fine tuning of reaction conditions and easy optimization, making the whole platform more flexible and productive. Lastly but most importantly, our cell-free strategy enables one-pot selective biotransformation by just adding different plasmids to construct relevant metabolic pathways in vitro . However, for cell-based production, laborious steps are needed to engineer strains and often one strain can only be used to produce one product without the property of selectivity as showcased with our cell-free systems. Taken together, our results highlight the flexibility of cell-free system for tunable and selective biotransformation with remarkable bioconversion efficiency. Looking forward, we envision that future efforts will continue to expand the types of biotransformation pathways and thus the products that can be reconstituted and synthesized in vitro .",
"introduction": "1 Introduction Environmental issues have attracted more and more attention, which are caused by the development of global industrialization and urbanization. Currently, municipal solid waste disposal has become a critical burden for urban development and kitchen food waste is a major component of municipal waste ( Ajay et al., 2021 ). Each year, about 1.6 billion tons of food waste are generated worldwide, which calls for advanced technologies to recycle and upgrade food waste into valuable products such as energy and materials ( Meng et al., 2022 ). The main compositions of food waste are carbohydrates, proteins, and lipids/oils; their proper disposal and/or recycling play an important role in global sustainable development ( Paritosh et al., 2017 ). Traditional solutions for the municipal waste treatment include landfilling and incineration, easily leading to seriously environmental issues such as air/soil pollution, greenhouse gas emission, and heavy metal leakage ( Hassan et al., 2020 ; Powell et al., 2016 ; Zhang et al., 2021 ). The use of food waste disposal units can decrease the amount of food waste; however, this method will aggravate the burden of water consumption and sewerage systems ( Iacovidou et al., 2012 ). Another solution is anaerobic digestion by microorganisms, which not only is environmentally friendly but also can produce bioenergy/biogas ( Badgett and Milbrandt, 2021 ). Yet, bioprocessing equipment and facilities are expensive and complex, which needs elaborate management hindering its wide application. In industry, long-chain hydrocarbon molecules have many applications in fragrances, cosmetics, lubricants, and biofuels, and thus possess great economic benefits ( Halfmann et al., 2014 ; Shi et al., 2018 ). Since food waste contains a large amount of long-chain fatty acids that can be derived from lipids/oils, using them as feedstocks for valuable product production might be a sustainable approach for the treatment of food waste. For instance, the compositions (%) of lauric acid in palm kernel oil and coconut oil are 47.8 and 46.5, respectively. Palmitic acid constitutes 44% of fatty acids in palm oil. In sunflower oil, the composition (%) of stearic acid is 4.5 ( Aransiola et al., 2014 ). While chemical methods have been established for fatty acid conversion, these processes often require harsh reaction conditions and rare-metal catalysts with low efficiency and poor selectivity ( Ford et al., 2012 ; Gomez et al., 2022 ; Witsuthammakul and Sooknoi, 2016 ). In biotechnology, different enzymes have been used to catalyze the bioconversion of fatty acids to value-added products including alkanes, fatty aldehydes, and fatty alcohols with high catalytic efficiency under mild reaction conditions. For example, fatty acid photodecarboxylase (CvFAP) found from the microalga Chlorella variabilis NC64A is able to convert fatty acids to alkanes (or alkenes), which can serve as biofuels, through decarboxylation initiated by blue light ( Sorigué et al., 2017 ). When CvFAP is coupled with lipase, triglycerides can also be converted to alkanes via a two-step cascade enzymatic reactions ( Huijbers et al., 2018 ). Another class of enzymes called carboxylic acid reductases (CARs) can catalyze the reduction of fatty acids to corresponding fatty aldehydes ( Butler and Kunjapur, 2020 ; Derrington et al., 2019 ). Note that CARs need to be post-translationally modified by an auxiliary enzyme phosphopantetheinyl transferase (PPTase) to form active holo -CARs ( Venkitasubramanian et al., 2007 ). Previous studies have shown that endogenous enzymes such as alcohol dehydrogenase (ADH) and aldehyde reductase (AHR) in E. coli are able to reduce a wide range of aldehydes (e.g., aromatic and aliphatic) to corresponding alcohols ( Akhtar et al., 2013 ; Derrington et al., 2019 ). As a result, heterologous expression of CARs in E. coli generates not only the target products (i.e., aldehydes) but also most often the side products (i.e., alcohols) due to the presence of many endogenous ADH/AHR enzymes. Deletion of specific ADH and/or AHR gene(s) from the host cell might minimize the aldehyde over-reduction. In contrast, if alcohols are final products, overexpression of ADH/AHR enzymes together with CARs is often a reasonable strategy as reported previously ( Butler and Kunjapur, 2020 ; Derrington et al., 2019 ). However, selective production/accumulation of aldehydes or alcohols using one type of engineered strain is difficult because the requirement of ADH/AHR enzymes is different. It is, therefore, necessary to develop a robust and simple approach for selective transformation of one substrate (e.g., fatty acid) to different target molecules as demanded in a single one-pot reaction. Recently, cell-free protein synthesis (CFPS) systems have been used for in vitro protein production and the construction of biomanufacturing factories ( Dudley et al., 2020 ; Ji et al., 2022 ; Lim and Kim, 2022 ; Liu et al., 2020 ; Rasor et al., 2021 ; Silverman et al., 2020 ; Xu et al., 2022 ). CFPS reaction mixture contains cell lysate, energy, amino acids, and salts, mimicking cell metabolism in vitro . By directly adding plasmid(s) to CFPS systems, cell-free production of desired products (e.g., proteins) can be achieved in hours without the use of intact living cells ( Silverman et al., 2020 ). Here, we propose to use the well-developed E. coli -based CFPS system for selective biotransformation of food waste (fatty acids) to long-chain hydrocarbons (e.g., alkanes, fatty aldehydes, and fatty alcohols) through controllable cell-free reaction conditions ( Fig. 1 ). Using cell-free system, we show two paradigms of selective bioconversions. One is reaction selectivity based on the added plasmids encoding different enzymes (here are CvFAP and CAR), which means that the same substrate can be converted to different products with different plasmid inputs. The other one is product selectivity in the CAR bioconversion system. By elevating the reaction temperature, the endogenous ADH/AHR enzymes originated from E. coli cell lysates can be deactivated so as to produce the intermediate fatty aldehydes catalyzed by CARs. Otherwise, ADH/AHR enzymes in the CFPS system will further reduce fatty aldehydes to fatty alcohols. Looking forward, we anticipate that cell-free biotransformation systems can be used for the rapid synthesis of fatty acid-derived high-value chemicals such as fatty aldehydes/alcohols and alkanes of industrial importance when selective biocatalysis in cells are difficult or not possible. Fig. 1 ( a ) Cell-free selective biotransformation of fatty acids to alkanes and fatty aldehydes/alcohols. Enzymes expressed with cell-free protein synthesis (CFPS) are used to construct in vitro metabolic pathways. ( b ) Enzymatic decarboxylation (top) and reduction (bottom) of fatty acids by CvFAP and CAR, respectively. Three fatty acid substrates used in this work are lauric acid (C 12 ), palmitic acid (C 16 ), and stearic acid (C 18 ). Fig. 1",
"discussion": "3 Results and discussion 3.1 Decarboxylation of fatty acids to alkanes CvFAP (a photoenzyme) was firstly discovered from a microalga in 2017, which can catalyze the decarboxylation of long-chain fatty acids to aliphatic hydrocarbons in response to blue light ( Sorigué et al., 2017 ). After that, its substrate scope and the enzyme activity have been extensively expanded through enzyme engineering and evolution ( Li et al., 2021 ; Xu et al., 2019 ; Zeng et al., 2021 ; Zhang et al., 2019 ). A previous study reported that the activity of purified CvFAP was lower than that of the crude enzyme solution prepared from the host E. coli cells ( Huijbers et al., 2018 ). While the reason remains unclear, it might be due to some unknown, yet necessary enzyme cofactors presented in the crude cell lysate. In this context, crude extract-based CFPS systems could be potential platforms to express CvFAP for efficient enzymatic catalysis without purification. Therefore, here we aim to use an E. coli -based CFPS system to express CvFAP that can directly catalyze the decarboxylation of fatty acids in situ . First, cell-free expression of CvFAP was carried out at 30 °C for 6 h. Then, the synthesis of CvFAP was confirmed by Western-blot analysis. The results indicated that CvFAP was successfully expressed with the correct molecular weight (71 kDa), although a small fraction of truncated proteins was formed ( Fig. S2a ). Having demonstrated the expression of CvFAP in CFPS, we next wanted to test the enzyme activity. For a positive reference, we also expressed CvFAP in E. coli ( Fig. S1b ) and prepared crude CvFAP enzyme solutions for the biocatalysis as reported previously ( Huijbers et al., 2018 ). To this end, we chose three fatty acids with different carbon-chain lengths (i.e., lauric acid, C 12 ; palmitic acid, C 16 ; and stearic acid, C 18 ) as substrates. The catalytic reactions were performed at 37 °C with gentle magnetic stirring under blue light (455–460 nm) illumination for 10 h ( Fig. S3 ). Note that if the reactions were not illuminated with blue light, no products could be detected ( Fig. S4 ). After reaction, the target products were extracted by ethyl acetate (adding 1 mM of 1-octanol as an internal standard) and then all samples were analyzed by GC-MS. The results suggested that all three fatty acids could be converted to their corresponding aliphatic hydrocarbons (alkanes) through decarboxylation by CFPS-expressed CvFAP ( Fig. 2 a and b, Fig. S5a ). We also tested substrate conversions of the three reaction groups over 10 h and observed that the enzymatic reactions basically stopped between 8 and 10 h ( Fig. S6 ). Moreover, we found that our CFPS system could achieve similar final conversions compared to the crude CvFAP enzyme solution-based biocatalysis in each substrate group ( Fig. 2 c and Fig. S6 ). The enzyme CvFAP particularly showed a high efficiency in our cell-free system toward palmitic acid (C 16 , 88% conversion) and stearic acid (C 18 , 95% conversion) ( Fig. 2 c). The conversion of lauric acid (C 12 ) was the lowest (13%) among the three tested fatty acids ( Fig. S5b ). Our finding is in agreement with previous reports that the substrates of C 16 and C 18 are more favored by CvFAP than C 12 ( Huijbers et al., 2018 ; Sorigué et al., 2017 ). Overall, our results demonstrate that cell-free system is feasible to express the photoenzyme CvFAP with catalytic activity. Since its discovery in 2017, CvFAP has been quickly employed for different enzymatic bioconversions ( Ge et al., 2022 ; Li et al., 2021 ; Xu et al., 2019 ; Zeng et al., 2021 ; Zhang et al., 2019 ). Therefore, we envision that CFPS will not only can be used for the construction of CvFAP-based metabolic pathway(s) to synthesize various products in vitro , but also may serve as a rapid method to engineer, express, and evaluate CvFAP variants if the design-build-test cycles performed in cells are laborious and time-consuming. Fig. 2 Decarboxylation of palmitic acid and stearic acid catalyzed by CvFAP. ( a ) GC-MS analysis of palmitic acid and pentadecane. ( b ) GC-MS analysis of stearic acid and heptadecane. ( c ) Substrate conversion (%) with cell-free expressed CvFAP and crude CvFAP enzyme solution. NC, negative control without plasmid in the reaction. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments. Fig. 2 3.2 Reduction of fatty acids by CARs 3.2.1 Demonstrating the activity of cell-free expressed CARs Having demonstrated the ability of using cell-free expressed CvFAP to perform in situ biocatalysis, we next sought to express CAR enzymes in CFPS to convert fatty acids to their reductive products. Typically, CARs are large multi-domain enzymes, consisting of an adenylation (A) domain for substrate activation, a thiolation (T) domain for substrate tethering and transferring, and a reductase (R) domain for substrate reduction ( Butler and Kunjapur, 2020 ; Derrington et al., 2019 ). Nascent apo -CARs are not active once freshly expressed and a post-translational modification is required to form functional holo -CARs by an auxiliary enzyme PPTase, which can transfer the phosphopantetheine group from coenzyme A (CoA) to a conserved serine residue in the T domain ( Venkitasubramanian et al., 2007 ). Normally, the promiscuous PPTase Sfp from B. subtilis ( Quadri et al., 1998 ) is used for post-modification of various CARs. After activation, holo -CARs can catalyze the reduction of carboxylic acids to corresponding aldehyde products. To reconstitute CAR-based bioconversion in vitro , we chose three enzymes: NiCAR ( He et al., 2004 ) and its variant NiCAR_Q283P with enhanced catalytic activity ( Schwendenwein et al., 2019 ), and AnCAR with high thermal stability ( Thomas et al., 2019 ). First, cell-free expression of the three CARs were confirmed by Western-blot analysis. The results showed that three CARs (∼129 kDa) were expressed with correct molecular weight bands, their expression levels were comparable, and all enzymes were almost completely soluble (see Western-blot in Fig. S2b ). Then, CARs were activated by Sfp to form functional ( holo ) enzymes. Given the flexibility of cell-free reactions, Sfp can be provided with purified Sfp or by coexpression of Sfp with CARs ( Fig. 3 a). To test the activity of CARs, palmitic acid (0.25 mM) was used as a substrate for the evaluation. After cell-free biotransformation, the samples were analyzed by GC-MS and two kinds of products (hexadecanal and hexadecanol) were observed (see Table S1 for their retention time). The results demonstrated that cell-free expressed CARs could be activated by Sfp to convert fatty acids to aldehyde products; however, aldehydes were further reduced to fatty alcohols by endogenous ADH/AHR enzymes. As shown in Fig. 3 b, three CARs were active but with different activities. The highest conversion (>40%) was observed in the group of using NiCAR_Q283P, which has been engineered with a higher activity compared to its parental NiCAR ( Schwendenwein et al., 2019 ). Moreover, we found that purified Sfp worked better than the coexpressed Sfp in all three CAR groups. This is similar to previous reports that purified Sfp can be directly added to cell-free systems to activate nonribosomal peptide synthetases ( Goering et al., 2017 ; Ji et al., 2022 ). Thus, purified Sfp was chosen to activate CARs during the following investigation and optimization. Fig. 3 Reduction of palmitic acid catalyzed by CAR. ( a ) Cell-free expression and activation of CAR for biocatalysis. ( b ) Effect of three CAR enzymes and Sfp (coexpressed or purified Sfp) on the substrate conversion (%). NC, negative control without plasmid in the reaction. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments. Fig. 3 3.2.2 Evaluation of substrate concentration on product formation Since the conversion (%) based on 0.25 mM of substrate (palmitic acid) was low ( Fig. 3 b), we next wanted to see if the product titer could be increased by adding higher substrate concentrations. Note that a previous optimized CFPS system is used in this work ( Jewett and Swartz, 2004 ; Kwon and Jewett, 2015 ), cell-free expression of CAR enzymes is not further optimized and their expression levels are comparable ( Fig. S2b , CFPS at 30 °C). Thus, we mainly focus on the effect of substrate concentration rather than optimization of enzyme expression/concentration for the biotransformation. To do this, we selected three fatty acid substrates (i.e., lauric acid, palmitic acid, and stearic acid) and increased their concentrations in cell-free reactions from 0.25 to 2 mM. Meanwhile, three CAR enzymes were tested for comparison. All cell-free reactions were carried out at 30 °C for a total of 16 h, including an initial 6 h for enzyme expression and another 10 h for subsequent biotransformation. Due to the presence of ADH/AHR enzymes in cell-free reactions, partial fatty aldehydes can be further reduced to fatty alcohols. Thus, the concentrations of the mixed products were determined together to calculate the substrate conversion (%). The results of palmitic acid bioconversion with NiCAR, NiCAR_Q283P, and AnCAR are shown in Fig. 4 . Clearly, NiCAR_Q283P performed the best and yielded the highest conversion (∼47%) at the lowest substrate concentration of 0.25 mM as compared to the other two enzymes (NiCAR and AnCAR). When the substrate concentration was doubled to 0.5 mM, the total titers of the mixed aldehyde and alcohol products (i.e., hexadecanal and hexadecanol) were also increased by nearly 2 times in all three CAR reaction groups. Further increases of substrate concentrations (>1 mM) did not improve the product titers, but reduced the total conversions in each group. This is probably due to the fact that the amount of enzyme expressed in CFPS is not changed and their ability for biocatalysis cannot be further improved regardless of increasing substrate concentration, thus leading to a lower conversion. Similar results were also observed for the other two substrates lauric acid and stearic acid ( Figs. S7 and S8 ). By considering both product titer and substrate conversion, we finally chose the substrate concentration of 0.5 mM for all bioconversion experiments in our following studies. Fig. 4 Effect of palmitic acid concentration on product formation catalyzed by ( a ) NiCAR, ( b ) NiCAR_Q283P, and ( c ) AnCAR, respectively. Final concentrations of the residual palmitic acid in each reaction group are summarized in Table S2 . Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments. Fig. 4 3.2.3 Enhancing the substrate conversion While cell-free expressed CAR enzymes were active toward three fatty acids ( Fig. 4 , Figs. S7 and S8 ), the overall conversion (%) of all tested substrates were relatively low. We next tried to test if an elevated reaction temperature could help improve the bioconversion. To this end, we ran cell-free reactions with different temperatures. The whole reaction process basically consisted of two stages: cell-free expression of CAR for 6 h and biocatalysis for product formation for another 10 h. For these two stages, we used four different temperature combinations, which are (i) 30 °C for both stages, (ii) 37 °C for both stages, (iii) 30 °C for CAR expression + 37 °C for bioconversion, and (iv) 37 °C for CAR expression + 30 °C for bioconversion. Note that in all cases, when the first reaction stage finished, the substrate was then added to the reaction mixture to start the second biocatalysis stage. The results indicated that different reaction temperature combinations did notably impact substrate conversions among all enzyme/substrate groups ( Fig. 5 , Figs. S9 and S10 ). The substrate palmitic acid was taken as an example ( Fig. 5 ). In each enzyme group, the highest conversion (%) was achieved with the same temperature combination, which is 30 °C for CAR expression and 37 °C for bioconversion. Remarkably, the conversion reached 92.7% under this condition (i.e., 30 °C + 37 °C) using the enzyme NiCAR_Q283P, which is > 2 times higher than that of the reaction with 30 °C for both stages ( Fig. 5 b). However, when both reaction stages were performed at 37 °C, the conversion was sharply reduced to 4.8% ( Fig. 5 b). Moreover, the substrate conversions from the groups of 37 °C + 30 °C were also as low as the groups of 37 °C + 37 °C ( Fig. 5 ). This is because the optimal temperature of the E. coli CFPS system is 30 °C and a high CFPS temperature at 37 °C leads to a low-level expression of CAR enzymes (see Fig. S2b for the comparison of CARs expression at 30 °C and 37 °C). As a result, under the best temperature combination, 30 °C can first support the expression of sufficient CAR and then cell-free expressed enzymes can catalyze the maximum conversion of the substrate at 37 °C, which is likely due to the fact that a higher temperature generally makes the enzymatic reactions going faster. Using the other two substrates lauric acid and stearic acid, we also observed similar trends of the total conversions under the four different reaction temperature combinations ( Figs. S9 and S10 ). Fig. 5 Evaluation of different reaction temperature combinations on the conversion of palmitic acid. ( a ) NiCAR. ( b ) NiCAR_Q283P. ( c ) AnCAR. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments. Fig. 5 3.2.4 Selective production of fatty aldehyde through thermal regulation Like cells, crude extract-based cell-free systems also contain endogenous ADH/AHR enzymes, leading to the over-reduction of fatty acids to alcohols. While it is challenging to control the selective transformation of fatty acids to aldehyde products in vivo , we propose that using cell-free systems might be a promising strategy to solve this issue. To address this opportunity, we next attempted to use a thermal regulation approach to deactivate ADHs/AHRs by heating to accumulate aldehydes rather than alcohol by-products in our cell-free reactions ( Fig. 6 a). To achieve the goal, we selected the thermostable AnCAR as a biocatalyst ( Thomas et al., 2019 ). After AnCAR was expressed, cell-free reactions were heated and then fatty acid substrates were added to the heat-treated mixtures to synthesize fatty aldehydes catalyzed by AnCAR. Note that the experiments with NiCAR and NiCAR_Q283P were also performed for comparison. To heat cell-free reaction mixtures, we incubated the reaction tubes in water bath for 5 min at temperatures ranging from 40 to 60 °C. Meanwhile, control experiments by heating at 30 °C were performed as well. Afterward, all biocatalysis reactions were carried out at 37 °C for 10 h, followed by detection of fatty aldehydes and corresponding alcohols using GC-MS. Fig. 6 Selective biotransformation of palmitic acid to hexadecanal. ( a ) Schematic diagram of cell-free selective bioconversion by thermal regulation. Accumulation of hexadecanal in cell-free reactions catalyzed by ( b ) NiCAR, ( c ) NiCAR_Q283P, and ( d ) AnCAR after heating deactivation of endogenous ADH/AHR enzymes. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments. Fig. 6 Overall, we found that as the heating temperature increased, the proportion (%) of fatty aldehydes in the product mixture (aldehydes and alcohols) significantly increased up to >90% ( Fig. 6 b, c, and d). It is clear that the thermostable AnCAR is more tolerant to heating than the other two enzymes (NiCAR and NiCAR_Q283P). When the reaction was heated over 50 °C, NiCAR and NiCAR_Q283P lost most of their activity. After heating at 50 °C, the product titers in the groups of NiCAR and NiCAR_Q283P significantly decreased by 80.5% and 83.3%, respectively, as compared to their control group (i.e., heating at 30 °C for 5 min) ( Fig. 6 b and c). By contrast, AnCAR could retain approximately half of its original activity after heating above 50 °C ( Fig. 6 d). In particular, when the reaction was heated at 55 °C, the proportion of the target product hexadecanal in the final product mixture reached 97.3%, which is nearly 2 times higher than that of the control group (50.6%) with heating at 30 °C ( Fig. 6 d). In addition to the substrate palmitic acid, we also observed similar results that AnCAR could catalyze lauric acid and stearic acid to lauraldehyde and octadecanal with a purity of 92.8% and 97.1%, respectively, after heat treatment at 55 °C ( Figs. S11 and S12 ). This is significant because we can selectively accumulate fatty aldehydes by deactivating thermal sensitive ADH/AHR enzymes with a simple heating strategy. However, we noticed that the final concentrations of hexadecanal maintained relatively stable in the reactions rather than further increased as hexadecanol decreased. Although AnCAR was reported as a thermostable enzyme, it lost its activity by 50% after heating at around 65 °C ( Thomas et al., 2019 ). That means high temperatures can still inactivate AnCAR. Here, after heating at 50–60 °C, the overall catalytic ability of AnCAR in the CFPS system was probably reduced to a similar level, which could only catalyze the formation of ∼50% product relative to the control ( Fig. 6 d). As a result, the accumulation of fatty aldehydes was not further enhanced even if the downstream reaction catalyzed by ADHs/AHRs was blocked. On the other hand, while it is not performed in the current work, we believe that one could improve the proportion (%) of fatty alcohols in the final product mixture by overexpression of ADHs/AHRs in the cell-free system, which can further drive the complete reduction of fatty aldehydes to corresponding alcohols. Therefore, our cell-free system enables easy product selectivity, which will provide a robust and flexible platform for selective biotransformation when cell-based in vivo systems remain difficult or not amenable."
} | 7,478 |
22028808 | PMC3197599 | pmc | 4,230 | {
"abstract": "In contrast to secondary succession, studies of terrestrial primary succession largely ignore the role of biotic interactions, other than plant facilitation and competition, despite the expectation that simplified interaction webs and propagule-dependent demographics may amplify the effects of consumers and mutualists. We investigated whether successional context determined the impact of consumers and mutualists by quantifying their effects on reproduction by the shrub Vaccinium membranaceum in primary and secondary successional sites at Mount St. Helens (Washington, USA), and used simulations to explore the effects of these interactions on colonization. Species interactions differed substantially between sites, and the combined effect of consumers and mutualists was much more strongly negative for primary successional plants. Because greater local control of propagule pressure is expected to increase successional rates, we evaluated the role of dispersal in the context of these interactions. Our simulations showed that even a small local seed source greatly increases population growth rates, thereby balancing strong consumer pressure. The prevalence of strong negative interactions in the primary successional site is a reminder that successional communities will not exhibit the distribution of interaction strengths characteristic of stable communities, and suggests the potential utility of modeling succession as the consequence of interaction strengths.",
"introduction": "Introduction The extreme intensity of the disturbance that results in primary succession is generally considered to be responsible for the differences in community assembly between primary and secondary succession. Ecologists have identified a variety of processes whose importance is magnified during primary succession, including amelioration of the physical environment, dispersal limitation, facilitative interactions and stochastic assembly [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] . In contrast, the effect of consumers on successional plant communities is regarded as more important in secondary succession [10] , [11] and in marine systems [12] , [13] , [14] , [15] . Similarly, because of their relative scarcity in primary succcesion, mutualists are also thought to be more important in secondary succession, and the ability to grow and reproduce without their aid is considered an important attribute of primary successional plant colonists [8] . Although interactions with consumers and mutualists are considered relatively unimportant for primary succession, a variety of studies indicate that they may strongly affect colonization of plant populations. For example, in early succession, consumers may temporarily escape their enemies and cause unusually large effects on plant population growth and spatial spread [15] , [16] , [17] , [18] . Likewise, the temporary absence of mutualists, such as pollinators (e.g., [19] , mycorrhizae [20] and nitrogen-fixing symbionts [e.g.] , [ 21,22] may disadvantage or temporarily exclude colonizing plant species that are dependent upon them. These studies suggest that the limiting effects of biotic interactions on colonizing plants can be greatly amplified during primary succession. This temporary inflation may be caused by successional properties of interaction webs. Primary successional sites, being the most intensely disturbed, generally have few species, low productivity, and support fewer trophic levels [23] , whereas secondary successional sites generally possess more complex sets of interacting species. Under these circumstances, consumers may anomalously impact a primary successional plant population, because secondary consumers or competitors that might weaken the interaction are temporarily lacking. These temporarily strong effects of biotic interactions may then translate to higher negative interaction strengths (i.e., effects on population dynamics) in primary successional communities. However, it is not yet known whether the impact of biotic interactions is more frequently promoted in the primary successional context, compared to the secondary successional context. The few existing examples focus on pairwise interactions, while studies of multispecies interaction remain particularly scarce [24] , [25] . More systematic examination of ensembles of consumers and mutualists across multiple successional contexts is required. The success of colonizing plant populations may be influenced by other factors from outside the local community. In particular, propagule pressure may form a key context early in succession, where the shift from immigration from external sites (donor control) to propagule production from within a site (local control) may strongly affect successional rates [15] , [26] . In general, the growth and spread of colonizing populations are highly dependent on propagule production and dispersal [e.g.] , [ 8] , [27,28] , and the effects of consumers or mutualists on seed production may be much larger relative to those in stable or declining populations [29] , [30] . In this study we asked three questions: 1) Does the limiting effect of multiple species interactions on plant reproduction vary with successional context? 2) Do consumers and mutualists differ in their influence on colonization during primary succession? and 3) How do successional differences in interactions combine with local control of propagule influx to affect colonization during primary succession? To address these questions, we investigated the effect of consumers (a fungal pathogen, pre- and post-dispersal seed predators, and an insect herbivore) and pollinator mutualists on black huckleberry ( Vaccinium membranaceum ) in primary successional and adjacent secondary successional areas created by the 1980 eruption of Mount St. Helens (Washington, USA).",
"discussion": "Discussion Our study finds that the effect of multiple species interactions on huckleberry depends on successional context, and provides support for the importance of biotic interactions during primary succession. The combined negative effect of consumers and insufficient pollination was greater for primary successional huckleberries (PS) than for secondary successional huckleberries (SS), and furthermore, these interactions have unusually large effects on PS populations. Our simulation models indicate that under primary successional conditions, the resulting seed loss should substantially diminish growth of the colonizing population. Our simulations also show that the dynamics of the colonizing population in primary successional habitat are strongly dependent on seed dispersal from both local survivors and the secondary successional population, counteracting seed loss and allowing for more rapid colonization. The effect of consumers and mutualists in succession The considerable difference between PS and SS may be attributed to pollinator and consumer behavioral responses to the sparse spatial distribution of huckleberry plants and other resources in PS compared to SS. The lack of effective pollination may be due to a shortage of pollinators, combined with the effect of geitonogamous or interspecific pollen transfer that often accompanies low plant density [e.g.] , [ 43,44] . It is worth mentioning that a complete absence of pollinators would result in no huckleberry reproduction, and no population growth without dispersal from external sources (e.g., no pollinators in Fig. 4 would produce a horizontal line), because huckleberry is largely self-incompatible. The greater risk of herbivory and seed predation to primary successional plants may have several related causes. Isolated plants may each have a larger “basin of attraction” for foraging insect seed predators. Rather than searching the surrounding primary successional landscape for scarce food sources, once a forager has located an isolated plant, it is likely to stay, possibly resulting in more consumers per plant [e.g., 45] . Insect consumers in primary successional areas are also more likely to escape their predators and thus reach higher population densities [e.g., 46] . Resource density in many areas of the Pumice Plain appears too low to support insectivorous vertebrate predators, such as birds and most small mammals, and low-density areas are depauperate in arthropod predators [29] , [47] , [48] . Our comparisons of simulated population growth demonstrated that insufficient pollination and reduced survival due to consumers combine to greatly limit population growth in PS. This result depends critically on the rate of seedling survival - at very low seedling survival (σ L <0.01), the number of seeds lost does not matter, while at higher seedling survival rates, removing the consumer interactions and increasing pollination rapidly increases recruitment to the adult class ( Fig. 4 , “no pollinator limitation, no consumers”). Releasing the population from consumer pressure, without supplementing pollination, allows for a somewhat less rapid increase in recruitment to the adult class as seedling survival increases ( Fig. 4 , “no consumers”). On the other hand, an increase in seedling survival does not appreciably increase population growth in the presence of consumers (“no pollinator limitation” and “control”). In other words, there is little effect of increasing pollination on population growth except in the absence of consumers. Thus, our simulation provides strong evidence that consumers, much more so than a shortage of mutualists, retard colonization of important later successional species at Mount St. Helens, and may therefore impact community trajectories. Vertebrate herbivores, such as moose and hares, can accelerate rates of primary succession in boreal flood plains and deglaciated sites [49] , [50] . In contrast, there are only a few examples of invertebrate herbivores impacting primary succession [16] , [47] . One of the best-documented cases is also from the Mount St. Helens Pumice Plain, where specialist insect herbivores decrease population growth rate and the rate of spread of alpine lupine ( Lupinus lepidus ) [17] , [18] , [47] , which decelerates succession at large spatial scales, but temporarily accelerates it at small scales by releasing lupine-held resources [51] , [52] , [53] . Succession and dispersal The strong effect of consumers within primary successional communities could be a general mechanism that contributes to slower species accumulation during primary succession compared to secondary succession. Certainly, without the mitigating effect of local control in our primary successional population, colonization would be extremely slow ( Fig. 5 ). The presence of biological legacies (remnant organisms and associated structures, living or dead) within a disturbed landscape is thought to be a major factor facilitating ecosystem recovery, especially after very large-scale disturbances [54] , [55] . Indeed, our simulations (and genetic analyses; [34] ) demonstrated a significant demographic impact of even a small number of survivors on the rate of colonization. Our results are in contrast to other studies, based on vegetation surveys of secondary successional refugia and surrounding primary successional sites at Mount St. Helens, which concluded that refugia facilitated invasion in the immediately adjacent areas of primary succession by vagile, pioneering species, but not by later successional species such as huckleberry [26] , [56] . These contrasting conclusions likely stem from the behavior of fruit-dispersing animals in primary successional habitat: frugivores that leave refugia do not linger in adjacent bare areas and thus seeds are dispersed long distances and deposited at low densities. Overall, we highlight the need for more studies of community assembly that include both successional stages, investigate the relative influence of local vs. donor control, and attempt to disentangle biotic and abiotic factors. Using matrix model simulations, we were able to explore how alternate sets of interactors and dispersal interact to influence rates of colonization. But, like many models, incomplete information limits our ability to make broader conclusions with our simulation model of colonization. As mentioned, the magnitude (but not relative importance) of species interaction effects on population growth rates depends on the level of recruitment ( Fig. 4 ), though the sparse recruitment rates that we have observed previously suggest that our estimated value is reasonable. In addition, the seed-to-seedling transition rate may not be constant over time, and may actually differ between PS and SS. Similarly, population growth rates will depend on other stages of the life cycle, though sensitivity analyses of these other life stages (see Figure S1 ) showed that our estimates do not affect conclusions regarding the relative impact of the interactions we focused on in this study. Although the population dynamics of woody plant species are not typically sensitive to early life stages [57] , our results suggest that seedling survival may actually be important for woody plants in nonequilibrium systems with temporal variation in the strength of consumer interactions. Biotic interactions and successional context We found that in primary succession the limiting effects of mutualists and consumers on colonizing plants were not only severe, but also amplified relative to their effect in more mature secondary succession. Our results also indicate that these reproductive effects can translate to substantial effects on population growth ( Figs. 4 and 5 ), and thus can be interpreted as interaction strengths (the effect of one species on the population growth of another, [58] ). The distribution of interaction strengths in early successional communities, and how that distribution is likely to change through successional time, are virtually unstudied. Empirical estimates from “non-successional” trophic webs repeatedly reveal a distribution characterized by a few strong interactions and many weak ones [25] , [58] , and models of consumer-resource interaction indicate that this skewed distribution confers stability [59] , [60] , [61] . Although not intended as models of succession (except [61] ), these models, together with empirical studies such as ours that document the occurrence of strong interactions affecting early plant colonists, suggest that successional communities in the early stages of assembly may exhibit more strong interactions than in later successional communities, or that they may lack other stabilizing properties that only occur in more diverse or successionally-advanced communities, such as nestedness [62] . The instability of young communities may produce radical shifts in the population dynamics of the existing interactors, which may directly change the distribution of interaction strengths via predator-prey time lags, extinction of predator or prey, or predator switching to more abundant prey. Alternatively, an unstable interaction strength distribution may continue until other species colonize and buffer the existing strong interactions with weaker interactions. For both of these scenarios, community assembly should continue if the unstable distribution of interaction strengths drives successional change. This new perspective on community succession calls for more studies that investigate the successional dependence of interaction strengths. Our study reveals an alternative view of succession, in which the distribution of interaction strengths shifts from a relatively high proportion of strong interactions toward distributions that are characteristic of stable communities. In particular, the combined effect of strong consumer pressure and absence of mutualists can significantly slow plant colonization, but this effect may be offset by local control of propagule influx. Although particular interactors may vary among systems, our study concludes that it is critical to consider the dependence of interaction strengths on successional context, and the potential importance of consumer and mutualist interactions for terrestrial primary succession."
} | 4,054 |
20463735 | null | s2 | 4,233 | {
"abstract": "Traditional robots rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment. A first step in this direction was the development of DNA walkers, which have developed from being non-autonomous to being capable of directed but brief motion on one-dimensional tracks. Here we demonstrate that previously developed random walkers-so-called molecular spiders that comprise a streptavidin molecule as an inert 'body' and three deoxyribozymes as catalytic 'legs'-show elementary robotic behaviour when interacting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as 'start', 'follow', 'turn' and 'stop'. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour."
} | 444 |
38257018 | PMC10818758 | pmc | 4,234 | {
"abstract": "Resistive switching memories are among the emerging next-generation technologies that are possible candidates for in-memory and neuromorphic computing. In this report, resistive memory-switching behavior in solution-processed trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) benzene–PVA-composite-based aryl acrylate on an ITO-coated PET device was studied. A sandwich configuration was selected, with silver (Ag) serving as a top contact and trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) benzene–PVA-composite-based aryl acrylate and ITO-PET serving as a bottom contact. The current–voltage (I–V) characteristics showed hysteresis behavior and non-zero crossing owing to voltages sweeping from positive to negative and vice versa. The results showed non-zero crossing in the devices’ current–voltage (I–V) characteristics due to the nanobattery effect or resistance, capacitive, and inductive effects. The device also displayed a negative differential resistance (NDR) effect. Non-volatile storage was feasible with non-zero crossing due to the exhibition of resistive switching behavior. The sweeping range was −10 V to +10 V. These devices had two distinct states: ‘ON’ and ‘OFF’. The ON/OFF ratios of the devices were 14 and 100 under stable operating conditions. The open-circuit voltages (Voc) and short-circuit currents (Isc) corresponding to memristor operation were explained. The DC endurance was stable. Ohmic conduction and direct tunneling mechanisms with traps explained the charge transport model governing the resistive switching behavior. This work gives insight into data storage in terms of a new conception of electronic devices based on facile and low-temperature processed material composites for emerging computational devices.",
"conclusion": "4. Conclusions In summary, we investigated the electrical characteristics of PVA film and a newly synthesized trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) benzene-based aryl acrylate (2-NVB)-PVA composite for non-volatile storage applications. They exhibited both resistive switching and the NDR effect. An investigation of the I–V characteristic displayed a hysteresis loop with non-zero crossing, showing our devices’ capacitive nature (extended memristor). They had good ON/OFF ratios of 14 and 100, with good endurance and retention properties. The charge transport model conformed to Ohmic conduction and filament conduction in the different regions of operation. Our work attempts to use a facile processed composite for flexible, foldable, and large-area applications.",
"introduction": "1. Introduction In the era of the More-than-Moore paradigm [ 1 ] and the Internet of Things (IoT), there is a requirement for new materials, device architectures, and technology. This is due to demands for portability, lightweight properties, bendability, stretchability, and ubiquitous applications. This demand evinces interest in flexible electronics. This has led to the development of conformal electronics for health care [ 2 , 3 ], flexible storage systems [ 3 ], display systems [ 4 , 5 ], and radio frequency identification systems [ 6 , 7 ]. One of the crucial things to realize about IoT gadgets is that they store information in non-volatile memory. Resistive memory devices [ 7 ] have become immensely popular among other emerging non-volatile-memory-like spin-transfer torque magneto-resistive (STT-MRAM) [ 8 ] and phase-change RAM (PCRAM) devices [ 9 ] over the past few decades. These devices have the advantages of being non-volatile, having fast access times, and having low power consumption [ 10 , 11 ]. Initially, resistive devices used chalcogenides and oxides [ 12 ]. With the saturation of the Moore model, there is now demand for new materials. A comparison of various emerging non-volatile memories is provided in Table 1 . Organic materials have provided another alternative for resistive switching memories. They are usually lightweight and flexible and have high data storage capacities and well-defined, simple layered structures making them suitable for resistive memory devices. Organic materials containing conjugated molecules and polymers have been researched for resistive switching memory applications. Small-component organic molecules like boron-based molecules (BF2BTDT) [ 13 ], pyrene-fused large N-heterocene (PyTTQ) [ 14 ], and benzothiadiazole-based molecules (NONIBTDT) [ 15 ] with structural tunability have shown resistive switching properties. In addition to single molecules, mixtures of organic molecules with two or three components have resulted in resistive switching behavior in MIM-type ITO/TCz:PDI/Al devices with tunable properties [ 16 ], and mixing a donor of indolo [3,2-b] carbazole (ICz) with a PDI acceptor (ICz:PDI = 2:1, 1:1, and 1:2) has resulted in binary to ternary properties [ 17 ]. Bozano and Yang et al. [ 18 , 19 ] have conducted a lot of work on incorporating metallic NPs into organic molecules to achieve the tunable characteristics of bistable memory. Potentially applicable metals are Al, Ag, Mg, Cr, and Ni [ 18 ]. They have shown an ON/OFF ratio of >10 4 , a high switching speed of <10 ns (delay), a switching endurance of >10 6 cycles, and data retention >10 5 s in multilevel switching devices. Single-component and multiple-layer polymers have optimized the macromolecule structure for bistable and multilevel resistive switching (RS). Some of the single-component materials include PVA composite [ 20 ], P55 [ 21 ], PMIDO3-based memory devices [ 22 ], and PFTPA-Fc [ 23 ]. They exhibit RS mechanisms via donor–acceptor (DA) charge transfer and conformational change mechanisms. Polymer blends of poly(methyl methacrylate) (PMMA) and poly(3-butylthiophene) (P3BT), serving as the active component [ 24 ], have been used. A Metal–Insulator–Metal (MIM)-type memory structure based on the st-PMMA/C60 complex has shown WORM-type switching characteristics [ 25 ]. Here, we are reporting the synthesis of trans, trans-1,4-bis-(2-(2-naphthyl)-2-(butoxycarbonyl)-vinyl) benzene (in short, 2-NVB) [ 26 ] and PVA composite as an active material for the resistive switching device. The I–V characteristics exhibited non-zero-crossing, nanobattery, capacitive, resistive, and inductive effects. The charge transport mechanisms and the process of the transfer of carriers between the interface and Ag ions were explored. A sandwich configuration of the M-I-M (Metal–Insulator–Metal) structure was considered. We were looking for an organic material for use as a dielectric for resistive memory devices. An analysis of the behavior was conducted for various transport models."
} | 1,655 |
21863016 | PMC3265375 | pmc | 4,236 | {
"abstract": "DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform.",
"discussion": "Discussion We have successfully applied nanomechanical DNA origami devices to construct visual 'single-molecule beacons' that can detect various targets of a wide range of molecular weights, from metal ions (a few tens of Da) to proteins (hundreds of kDa), at molecular resolution using the same platform. Each of the detection mechanisms we have described—pinching, zipping, or unzipping—for the targets of interest can be freely chosen and used orthogonally on differently shaped origami devices in a single mixture. Comparison between the zipping experiments with Na + and TeloE ( Fig. 4c ) and preclosure of miR20-triggered unzipping systems ( Supplementary Fig. S3 ) may hint at the border between targets appropriate for pinching and those require zipping. When only one zipper-element pair was introduced into DNA pliers, Na + with one TeloE gave ca. 40% closure of DNA pliers. In contrast, the yield of preclosed DNA pliers with only one preclosing zipper-elements pair was as low as 15% (whereas the parallel fraction without any zipper element was around 6%). Increasing the number of zipper-element pairs to two resulted in almost the same yield, around 50%, for both systems. Thus, the border should not be far from these zipper elements. The melting temperature of the TeloE dimer in the presence of 100 mM Na + is 42 °C, whereas the melting temperature for the 10-bp complementary part of the miR20 targeting elements is 31 °C under the conditions employed in the study (12.5 mM Mg 2+ and 4 nM strand concentration, data not shown) or Δ G ° 37 =−11.04 kcal mol −1 (1 M Na + ) 42 . The maximum yield of parallel form in a solution containing a single kind of DNA origami device was ~80%, even for the preclosed DNA pliers used in unzipping systems, which were directly annealed into the closed form whereas the whole lever structure was in the folding process ( Supplementary Fig. S3 ). This maximum yield is rather low compared with the ~90% folding yield reported for simple rectangular DNA origami 18 , but it is still high considering the relatively small number of linkages, including an unfavourable parallel four-way junction, joining the two levers. The observation of origami devices using AFM is a completely single-molecule method. Therefore, the theoretical detection limit of the systems should be exceedingly small, if the reaction volume is further reduced with the aid of microfluidics and MEMS technology. For example, a 4nM solution of DNA origami devices, a typical condition used in the present study, corresponds to 1 origami device molecule in every 0.4 femtolitre (=μm 3 ), which contains 2 target molecules for SA and IgG detection, 50 molecules for 200 nM miRNA, or 2,500 atoms for 10 μM Ag + . Moreover, almost any kind of protein or small molecule can be a target of DNA origami devices by inverting the polarity of the pinching systems from an antigen-modified origami device/antibody combination to an antibody-modified origami device/antigen combination. Taking advantage of the rather large size of the origami devices, the capture and detection of whole virus capsids is feasible too 43 . The development of allosteric metaenzymes by attachment of another functional nanomaterial such as an enzyme to the other end of the levers, enabling the switching of their activity by mechanical movement, would be another interesting application of DNA pliers. Such metaenzymes may provide an extra detection pathway for the structural changes of DNA origami devices: chemical signal amplification, which is popularly employed today, for example, in enzyme immunoassays. The present system may be a first step toward powerful tools in future studies of various nano-biochemical interactions."
} | 1,179 |
27279149 | PMC4899792 | pmc | 4,237 | {
"abstract": "Silk has attracted widespread attention due to its superlative material properties and promising applications. However, the determinants behind the variations in material properties among different types of silk are not well understood. We analysed the physical properties of silk samples from a variety of silkmoth cocoons, including domesticated Bombyx mori varieties and several species from Saturniidae. Tensile deformation tests, thermal analyses, and investigations on crystalline structure and orientation of the fibres were performed. The results showed that saturniid silks produce more highly-defined structural transitions compared to B. mori , as seen in the yielding and strain hardening events during tensile deformation and in the changes observed during thermal analyses. These observations were analysed in terms of the constituent fibroin sequences, which in B. mori are predicted to produce heterogeneous structures, whereas the strictly modular repeats of the saturniid sequences are hypothesized to produce structures that respond in a concerted manner. Within saturniid fibroins, thermal stability was found to correlate with the abundance of poly-alanine residues, whereas differences in fibre extensibility can be related to varying ratios of GG X motifs versus bulky hydrophobic residues in the amorphous phase.",
"discussion": "Discussion The main objective of this study was to correlate the physical properties of diverse silkworm silks with the amino acid sequences of the underlying fibroins. Based on phylogeny, the silks studied belong to two main groups: the bombycoid type (represented by the B. mori varieties) and the saturniid type (the rest of the samples), which exhibited significant differences in the fibroin repetitive sequences at different levels of organization. The repetitive blocks in B. mori fibroin feature long concatenated blocks of (GA) n G X of varying lengths and arrangements, with GAGAGS repeats constituting the intermolecular β-sheets of the crystalline fraction, while the numerous Tyr residues in GAGAGY repeats are predicted to occupy semi-crystalline regions whose conformation has not yet been well resolved 50 . Due to the variability among the concatenated blocks, the sequence correspondence between laterally adjacent fibroin chains is not expected to persist over long distances, thus tending to produce heterogeneous crystalline units along the fibre. This is consistent with the large spread and general lack of consensus regarding the crystallite dimensions of B. mori silk 50 51 . The complexity inherent in the B. mori fibroin structure may explain the tensile test results. In particular, the gradual yielding during fibre extension ( Fig. 2a ) may be attributed to the non-uniform distribution of stress among the heterogeneous crystalline structures. Likewise, the relative lack of distinct features during thermal analyses could reflect a lack of concerted changes in the polymer conformations as a consequence of the heterogeneous fibre structure ( Figs 3 and 4 ). In contrast, the most prominent feature of the saturniid fibroin sequences is the strictly repetitive nature of the poly(Ala) and non-poly(Ala) blocks. We hypothesize that the well-defined transitions during the tensile deformation of saturniid silks reflect coordinated molecular motion that may be attributed to the high degree of modularity in the primary structures. Similarly, the sharp boundaries and well-defined endothermic peaks observed during thermal analyses (particularly in Antheraea and Samia ) might be indicative of concerted effects within the fibroin chains caused by heating. It is useful to compare the saturniid sequences with spider dragline silk (major ampullate silk), whose constituent proteins (spidroins) bear strikingly similar set of amino acid motifs, likewise arranged as alternating poly(Ala) and Gly-rich repeats ( Fig. 6a ). Dragline silk has been the subject of numerous investigations, and hypotheses regarding its molecular structure and function have been developed to a much greater extent compared to silkworm silks 52 53 . At the nanoscale, dragline silk features well-ordered crystallites (stacked β-sheets) embedded in a matrix of amorphous chains, corresponding to the ordered and disordered fractions of the silk polymers, respectively 54 55 . Similar to saturniid silks, the tensile deformation of dragline silk produces prominent yielding and post-yield strain hardening 11 56 . The yield point has been equated with a glass-to-rubber transition within the amorphous regions 57 , and post-yield strain hardening to the reconversion of the rubber states back to glass or crystalline states at higher elongation 53 . Among the silkworm sequences investigated here, S. c. ricini fibroin bears the closest resemblance to dragline spidroin, MaSp1, with all tandem repeat subtypes being rich in GG X motifs 58 ; interestingly, the qualitative stress-strain profiles of the two silk types are remarkably similar 56 . It should be noted that dragline silk also includes a second spidroin component, MaSp2, whose proline-rich sequence has been linked to the extraordinary ductility of dragline silk 59 . The three wild silks investigated ( R. fugax , A. aliena , and S. jonasii ) displayed inferior extensibility in tensile testing and relatively ill-defined transitions during thermal analyses. The corresponding fibroin sequences from these species feature a higher abundance of large hydrophobic residues within the non-poly(Ala) blocks at the expense of the GG X motifs compared to the other saturniids ( Table 2 ). These bulky groups might form hydrophobic aggregates within the amorphous phase, that when combined with a low abundance of glycine residues could account for the decreased ductility (increased brittleness) in these silks. However, the biological significance of these differences in fibre properties, with respect to in vivo cocoon function, remains unknown. In this study we have sought to examine the extent to which the differences in the primary structures of the fibroin molecules from different lineages of silk moths influence their material properties. Based on our results, certain relationships could be observed, for instance between the overall thermal stability of the silk fibres and the proportions of crystallite forming residues (poly-alanine) in the case of saturniid fibroins, or the correlation between brittle silk types and the preponderance of bulky hydrophobic residues within the repetitive sequences. Thus in certain respects the sequence of the constituent fibroins is a major determinant of the observable physical characteristics. However, in terms of tensile properties the overall picture is more complex. Although silk fibres from the two main lineages investigated typically follow different deformation paths that can be related to the differences in modular organization of their repetitive sequences, individual samples exhibited a wide degree of variability in their measured tensile parameters. Indeed, although the amino acid sequence of the constituent fibroin undoubtedly plays an important role in shaping material properties, other factors such as the spinning process 25 26 , reeling rate 60 , fibre morphology 36 61 , flaw distribution 30 , temperature 62 , humidity 63 64 and degree of sericin binding 65 have also been shown to modulate the mechanical properties of silk fibres. There is doubtless a subtle and complex interplay between these different intrinsic and extrinsic factors."
} | 1,886 |
25797008 | null | s2 | 4,239 | {
"abstract": "The genomics and proteomics revolutions have been enormously successful in providing crucial \"parts lists\" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The \"sum-of-the-parts\" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture."
} | 424 |
34211966 | PMC8239229 | pmc | 4,240 | {
"abstract": "To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO 2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.",
"conclusion": "Conclusion and Future Directions Computational protein engineering promises a fast and efficient identification of enzyme variants with altered properties tailored for sustainable biomanufacturing of chemicals compared to experimental engineering techniques. The need for stability, specificity, and activity optimized biocatalysts drives the improvement of software along the engineering pipelines presented here. Still, proof of concept for the utilization of generalizable engineering pipelines including enzyme structure prediction, design, screening, and characterization of enzyme variants in silico is missing. The Rosetta modeling suite included in webserver applications and local software packages presents the option to perform and automate most tasks required in computational enzyme engineering by only one software. Implementation of automated molecular docking and screening platforms in webserver applications such as FuncLib could further increase the performance of Rosetta-based sequence design. The continuously increasing accuracy of protein structures prediction by machine learning–based approaches propels the development of fast and precise enzyme modeling and engineering pipelines to complement experimental engineering methodology or even open up the optimization of biocatalysts for which experimental techniques are missing. Complementing computational enzyme engineering with MD simulations provides powerful means to understand how mutations are affecting substrate binding and enzymatic activity. In the future, such computational approaches will accelerate the discovery of optimized protein-based solutions in general and of biocatalyst in particular for biotechnological and biomedical application. The computational pipelines described here can help to overcome the experimental limitations in metabolic pathway optimization on a protein level to enable, for example, industrial scale production of biofuels.",
"introduction": "Introduction At the start of the third decade of the twenty-first century, humankind faces a multitude of challenges regarding climate change ( Arnell et al., 2019 ), air pollution ( Wang et al., 2019 ), and a shrinking number of intact ecosystems ( Nolan et al., 2018 ) due to human activity. The demand for sustainable solutions addressing the basis of chemical production, transport, and agriculture to enable a net zero-carbon society is higher than ever before ( Hoegh-Guldberg et al., 2019 ). Hence, the development of technologies substituting fossil resources is an important goal of current scientific research ( Xu et al., 2018 ). Utilizing the synthetic power of microorganisms for the sustainable production of bulk chemicals and fuels to replace chemicals currently generated from fossil fuels and tropical plant agriculture is an important contributor toward the goal of achieving a net zero-carbon society ( Rodionova et al., 2017 ). In this regard, biosynthesis of hydrocarbons in microorganisms can be a sustainable technological alternative to produce fuels for aviation ( Schirmer et al., 2010 ; Kallio et al., 2014a ), a model of transportation for which competitive electric solutions are still missing ( Schäfer et al., 2019 ). The benefits of applying biocatalysts in the industrial production of commodity chemicals compared to inorganic catalysts lie mostly in their ability to facilitate enantioselective conversions at ambient conditions (temperature and pressure) ( Woodley, 2020 ). Additionally, the usage of biocatalysts instead of metal catalysts, for example, can reduce the amount of waste of chemical production because biocatalysts can be recycled easily ( Sheldon and Woodley, 2018 ). Further optimization of biocatalysts can expand the solution space of an enzyme and enable the identification of novel synthetic pathways for biomanufacturing of chemicals ( Erb et al., 2017 ). Hence, engineering enzymes for tailored substrate specificity ( Amer et al., 2020 ; Eser et al., 2020 ), catalytic efficiency ( Risso et al., 2020 ), and stability ( Goldenzweig et al., 2016 ; Yu et al., 2017 ) are important for the implementation of novel biosynthetic systems. Efforts for enzyme engineering are exponentially growing due to the demand for natural or biologically produced chemical compounds, such as alcohols, hormones, or essential oils, either by constructing de novo –designed pathways or by optimizing existing ones ( Marcheschi et al., 2013 ). In addition, current progress in genome sequencing identified a number of new enzymes or strain-specific variants that may be an alternative for the application in biotechnology; however, in a lot of cases, they are not stable or suitable for standard expression strains. Currently, enzyme engineering efforts are mostly based on rational engineering with low- and medium-throughput screening of small libraries ( Figure 1A ) and directed evolution-based approaches and high- and ultrahigh-throughput screening ( Figure 1B ; Ma et al., 2021 ); nevertheless, also de novo approaches start to get more attention and had been already used in several works ( DeLoache et al., 2015 ; Dou et al., 2018 ). Interestingly, including computational tools ( Romero-Rivera et al., 2017 ) as evolutionary conservation analysis ( Ashkenazy et al., 2016 ), mutant structure modeling ( Khersonsky et al., 2018 ; Leman et al., 2020 ), and molecular dynamics (MD) simulations ( Yu and Dalby, 2018 ; Surpeta et al., 2020 ) is becoming more abundant and has the potential to accelerate the identification of highly stable and productive biocatalysts for sustainable application ( Figure 1C ). The development of easy-to-use software and tools available as online servers makes it possible for researchers who are not experts in computational biology to apply state-of-the-art computational protein engineering methodology. On the other hand, the data from in silico engineering do not necessarily correlate with experimental data ( Pucci and Rooman, 2016 ; Carlin et al., 2017 ), and thus more advanced pipelines using multiple computational tools are required for accurate mutant structure modeling and energy predictions. The application of engineered proteins is versatile and covering various technological branches from pharmaceutics to bioelectronic devices ( Kalyoncu et al., 2017 ) and biosensors ( Xiong et al., 2017 ; Kunjapur and Prather, 2019 ). FIGURE 1 Experimental protein engineering strategies and an idealized scheme for a design–test–build–learn cycle of optimizing enzymes using computation. (A) Exemplified workflow for low- and medium-throughput enzyme engineering strategies. (B) Exemplified workflow for high- and ultrahigh-throughput enzyme engineering strategies. (C) Design–test–build–learn cycle for industrial chemical production including computational methodology and production–consumption–recycling cycle of chemical usage. Promising enzyme variants are identified computationally, which leads to targeted experimental testing. The metabolic systems are then applied in microorganisms for industrial-scale production. The experimental implementation provides additional information for computational optimization. Consumption of chemicals as biofuels results in the release of CO 2 , which can be recycled by microorganisms in bioreactors to close the cycle. Our research in synthetic biology and metabolic engineering is directed toward developing methods for bioproduction of renewable chemicals with special emphasis on biofuel-producing pathways. We and others have found that conventional strategies such as directed evolution are not applicable to all enzymatic reactions for lack of high-throughput assays that are required for the effective use of laboratory-evolution strategies. This has turned our attention to computational enzyme engineering methodology that can guide the experimental efforts. It is important to note that the modules described here can be applied with necessary adjustments to all kinds of protein engineering tasks and are therefore not limited to the field of metabolic enzyme engineering. Still, the application of computational methodology will be discussed on the example of metabolic enzymes involved in biofuel production to highlight strengths and limitations of such approaches on a particular field of biotechnological research. In this article, we review computational tools that can be used to create a platform for fast and customizable modeling and evaluation of promising enzyme variants in silico ( Figure 2 ). We focus on methods that have been experimentally validated and shown to outperform conventional in vitro selection methods. We conclude that computational enzyme engineering can accelerate the development of synthetic metabolic pathways for industrial use. FIGURE 2 Computational enzyme engineering pipelines. Module 1: structure–function analysis to identify active site and substrate-binding pocket. Module 2: building enzyme–substrate complexes with molecular docking approaches. Module 3: identification of design positions for the subsequent sequence design. Module 4: engineering stability of enzymes with PROSS and FireProt. Module 5: engineering activity and specificity of enzymes with FuncLib, IPRO, CADEE, and HotSpotWizard. Module 6: screening for stability, affinity, and activity changes with DUET, STRUM, KDEEP, and mCSM-lig."
} | 2,653 |
31937786 | PMC6959354 | pmc | 4,242 | {
"abstract": "Glucose and xylose are the major components of lignocellulose. Effective utilization of both sugars can improve the efficiency of bioproduction. Here, we report a method termed parallel metabolic pathway engineering (PMPE) for producing shikimate pathway derivatives from glucose–xylose co-substrate. In this method, we seek to use glucose mainly for target chemical production, and xylose for supplying essential metabolites for cell growth. Glycolysis and the pentose phosphate pathway are completely separated from the tricarboxylic acid (TCA) cycle. To recover cell growth, we introduce a xylose catabolic pathway that directly flows into the TCA cycle. As a result, we can produce 4.09 g L −1 \n cis , cis -muconic acid using the PMPE Escherichia coli strain with high yield (0.31 g g −1 of glucose) and produce l -tyrosine with 64% of the theoretical yield. The PMPE strategy can contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.",
"introduction": "Introduction Research on the microbial production of useful materials has gained interest and attention as an alternative production method of petrochemical products 1 – 3 . Metabolic engineering has made great contributions to advances in bioproduction 4 – 6 on the premise of using only particular sugars as carbon sources 7 – 10 . Lignocellulosic biomass, which does not compete with global food supplies, is a promising raw material for bioproduction 11 , 12 . Glucose, existing as a component of cellulose, is the most common monosaccharide obtained from woody biomass 13 . Hemicelluloses, which contain xylose as the main constituent, represent ~20–40% of lignocellulosic biomass 14 . The co-utilization of glucose and xylose in lignocellulosic biomass is essential for the economically feasible production of biofuels and chemicals 15 – 17 . One problem with the use of mixed sugars is carbon catabolite repression (CCR) 18 , 19 . When E. coli is cultivated with glucose–xylose mixtures, glucose is preferentially consumed before xylose because of CCR, which decreases the production rate and/or titer; thus, it is necessary to repress or avoid CCR to efficiently utilize glucose–xylose mixtures 20 . A successful strategy for relieving CCR is the knockdown of the phosphotransferase system (PTS) and the application of adaptive evolution to improve sugar co-utilization 21 . Disrupting l -arabinose transcriptional regulator, which acts as a repressor of xylose catabolite enzymes, is another strategy to eliminate CCR 22 . Wang et al. engineered an E. coli strain that utilized glucose–xylose mixtures for methyl ketone production 23 . To inhibit CCR, glucose uptake was reduced by disrupting the gene encoding glucose-specific PTS enzyme II component ( ptsG ), and xylose uptake was enhanced by the expression of a xylose transporter and xylose isomerase, which are encoded by xylA and xylF , respectively. This simple modification of metabolism allowed the strain to simultaneously use both the sugars, but it caused decreases in productivity and yield. The two-strain co-culture system is another method for efficiently utilizing glucose–xylose mixtures 24 , 25 . Zhang et al. used a co-culture system with two metabolically engineered strains to increase the production of cis,cis -muconic acid (MA) 25 . MA, a shikimate pathway derivative, is a valuable compound that is a precursor of important chemical compounds, such as adipic acid and terephthalic acid, and its production has recently attracted attention 7 , 26 – 30 . The glucose-consuming strain synthesized 3-dehydroshikimic acid (DHS) and secreted it in culture medium, whereas the other xylose-consuming strain converted DHS to MA. Co-culture using strains assimilating each specific sugar resulted in high production of MA from mixed sugars, thus successfully avoiding CCR. However, in fermentation using a two-strain co-culture, several problems, such as complicated operations and difficult handling arise. In bioproduction, another common problem is that carbon leaks into pathways other than the target pathway (Fig. 1a ) 31 . Phosphoenolpyruvate (PEP), pyruvate (PYR), and acetyl-CoA are important intermediates of target chemicals as well as tricarboxylic acid cycle (TCA cycle) components 4 . Hence, the biosynthetic pathways of chemicals derived from PEP, PYR, and acetyl-CoA compete for carbon flux with the TCA cycle, leading to decreased product yields 32 . However, the disruption of the TCA cycle or carbon flux into the cycle results in metabolic imbalance and negative effects on cell growth 33 – 35 . This means that target chemical production and cell growth are in an inverse relationship. As one solution, a switching system named metabolic toggle switch between the TCA cycle and target chemical production pathway was developed 32 . To toggle between cell growth and production, the expression of enzymes related to acetyl-CoA metabolism ( gltA ; citrate synthase) was controlled by transcriptional regulation. Using this system, isopropanol production was improved by as much as 3.7 times relative to that in the parental strain level. Although metabolic toggle switching is a useful tool for redirecting metabolic pathways, the ON/OFF timing of toggle switches (i.e., adding an inducer to the culture) needs to be optimized. Fig. 1 The concept of parallel metabolic pathway engineering. a Glucose and xylose carbon fluxes in common strains. Glucose and xylose are catabolized by the same pathways, glycolysis and the pentose phosphate pathway, and a large amount of the liberated carbon is used to synthesize biomass constituents and produce energy. b Glucose and xylose carbon fluxes in the engineered parallel metabolic pathway strain. Metabolic pathways of glucose and xylose do not intersect, making them metabolically parallel. Blue, yellow, and green arrows represent carbon flow from glucose, xylose, and both, respectively. The red-bordered yellow arrow represents the introduced exogenous xylose catabolite pathway. The thickness of arrows represents the proportion of carbon flow. Red crosses and dotted lines indicate the disruption of metabolic pathways. In this study, we propose parallel metabolic pathway engineering (PMPE) for co-utilizing glucose–xylose without decreasing growth ability. Glycolysis and the pentose phosphate pathway (PPP) are completely separated from the TCA cycle, and carbon supply from these pathways (i.e., PEP, PYR, and acetyl-CoA) is completely blocked from the TCA cycle. To recover cell growth (i.e., supply TCA cycle intermediates), a xylose catabolic pathway that directly flows into the TCA cycle without interfering glycolysis and PPP is introduced (Fig. 1 ). As a result, glucose is used mainly for target chemical production, and xylose is only used for cell growth. PEP is the most important precursor derived from glucose via glycolysis for chemical production. PEP is also a starting metabolite of the shikimate pathway as well as a precursor of the TCA cycle. Shikimate pathway-derived chemicals such as MA are among the most suitable targets for PMPE strains because they cause no carbon leaks from glycolysis into the TCA cycle. The highest yields of MA reported till date are 0.21 and 0.35 g g −1 of glucose in batch culture and complicated fed-batch culture, respectively 25 , 26 , 29 . Although some researchers partially disrupted the pathways connecting glycolysis and PPP to the TCA cycle to prevent carbon loss from the TCA cycle 25 , 29 , 36 . there are no reports in which these pathways were completely removed. In many organisms, xylose is catabolized by the isomerase and oxo -reductive pathways into d -xylose 5-phosphate, which then enters PPP 37 . To construct a parallel metabolic pathway (PMP), we focused on the Dahms pathway. Caulobacter crescentus , an oligotrophic bacterium, catabolizes xylose via an alternate pathway 38 . The Dahms pathway in C. crescentus directly produces PYR and glyoxylate from xylose without glycolysis and PPP (Supplementary Fig. 1 ). In the Dahms pathways, each reaction step has a higher negative change of Gibbs energy than the isomerase and oxo -reductive pathways, which indicates high thermodynamic favorability 37 . Chemical production using the Dahms pathway has been successfully demonstrated with high yields 39 , 40 ; however, there is no report regarding the use of this pathway for cell growth and maintenance. We assume that the Dahms pathway, which can provide PYR and glyoxylate from xylose as a carbon source, will be suitable for growth recovery in cells in which glycolysis and the TCA cycle cannot supply the necessary components. To prove our concept, we produce MA using the PMPE E. coli strain. We modify the metabolic pathway of E. coli so that only glucose could be used for MA production, and we introduce the Dahms pathway to restore cell growth. We efficiently produce MA from glucose–xylose mixtures and investigate the fractional contribution of these sugars via 13 C-metabolic analysis. To confirm the versatility of PMPE, we demonstrate the production of l -tyrosine which is another shikimate pathway derivative and 1,2-propanediol.\n\nIntroducing the Dahms pathway leads to growth on glucose The exogenous xylose catabolic pathway for recovering the cell growth of the CFT5 strain needs to have the following two features: (i) ability to synthesize the essential metabolites of the TCA cycle from xylose and (ii) independence from both glycolysis and PPP. We focused on the Dahms pathway that synthesizes PYR and glyoxylate from xylose without using glycolysis/PPP-related enzymes. In the Dahms pathway, xylose dehydrogenase ( xdh ) from C. crescentus converts xylose to xylonolactone. Xylonolactonase ( xylC ) from C. crescentus converts xylonolactone to xylonate. Xylonate is converted to 2-dehydro-3-deoxy- d -xylonate (DHDOX) by endogenous xylonate dehydratase ( yjhG ), and then DHDOX is converted to PYR and glycolaldehyde by endogenous DHDOX aldolase ( yjhH ) (Supplementary Fig. 1 ). Glycolaldehyde is metabolized to glyoxylate, after which it enters the TCA cycle. A strain, CFT5x, harboring xdh , xylC , and an endogenous yjhHG expression plasmid (Fig. 2a ), was cultured in M9 minimal medium with glucose, xylose, or a glucose–xylose mixture. When xylose-containing medium was used, CFT5x did not grow (Fig. 2b, c ), suggesting that supplying metabolites synthesized from glycolysis or PPP was not sufficient for growth using xylose as the sole carbon source. Unexpectedly, CFT5x grew in M9 minimal medium containing glucose as the sole carbon source, suggesting that introducing the Dahms pathway may activate carbon flow into the TCA cycle from glucose but not xylose. We examined the effect on the cell growth by expressing each enzyme of Dahms pathway, Xdh, CcxylC, and YjhHG in CFT5. As a result, only the strain expressing Xdh could grow in M9 minimal medium containing glucose as the sole carbon source (Supplementary Fig. 3 ). However, Xdh has no or little enzymatic activity for glucose or glucose-6-phosphate as substrates 38 . Thus, 6-phosphogluconate is one of the possible candidates. Fig. 2 Strain construction and cell growth examination. a Metabolic design of CFT5x. b Bacterial cell growth. Blue, black, and red symbols indicate the growth of the CFT5x strain and ATCC31882 cultured in medium supplemented with glucose, a glucose–xylose mixture, and xylose, respectively. c Glucose and xylose consumption. Filled blue symbols indicate glucose consumption in glucose-containing medium. Filled red symbols indicate xylose consumption in xylose-containing medium. Open blue and open red symbols indicate glucose and xylose consumptions in mixed sugar-containing medium, respectively. G6P glucose-6-phosphate; PEP phosphoenolpyruvate; E4P erythrose 4-phosphate; DHS dehydroshikimate; and Gluconate-6P gluconate-6-phosphate. The data are presented as the average of three independent experiments, and error bars indicate standard errors. Source data underlying Fig. 2b, c are provided as a Source Data file.\n\nGrowth of the GX1x strain by introducing the Dahms pathway To regain cell growth, we introduced the Dahms pathway into the GX1 strain. GX1x strain equipped with the Dahms pathway (Fig. 4a ) grew in M9 minimal medium with a glucose–xylose mixture as the carbon source, and maximum OD 600 reached at the same level of ATCC31882 (Fig. 4b ). Conversely, the strain did not grow in media containing glucose or xylose alone. Carbon from glucose could not flow into the TCA cycle, which leads to deficiencies of the essential metabolites and energy necessary for cell growth. In the medium containing glucose–xylose mixture, the Dahms pathway supplied PYR and glyoxylate from xylose as carbon sources, thereby restoring cell growth. It was thought that the GX1x strain did not grow in M9 minimal medium containing only xylose because of the insufficient activity of the endogenous xylose catabolic pathway (i.e., xylAB ). We constructed the strain overexpressing XylA and XylB derived from GX1x. Contrary to expectations, this strain did not grow in M9 minimal medium with xylose as a sole carbon source. Furthermore, the strains derived from CFT5 overexpressing Dahms pathway enzymes (Xdh, CcxylC, YjhH, and YjhG) and/or enzymes of the endogenous xylose catabolic pathway (XylA and XylB) also did not grow in the same condition (Supplementary Fig. 6 ). In this strain, while PYR and glyoxylate were supplied via Dahms pathway, these metabolites would have been converted to intermediates of glycolysis and could not be used in the TCA cycle. Additionally, we analyzed the metabolome in PMPE strain. As a result, PMPE strain considerably accumulated PEP and had been enhanced with PPP. These phenotypes are suited for the production of shikimate pathway derivatives (Supplementary Discussion 2 , Supplementary Figs. 14 , 15 ). Fig. 4 Examination of the growth of the strain in which the Dahms pathway was introduced. a Metabolic design of the GX1x strain. b Bacterial cell growth in minimal medium. Gray, blue, and orange symbols indicate the growth of the GX1x strain cultured in medium containing only glucose, a glucose–xylose mixture, and xylose alone, respectively. Yellow symbols indicate the growth of the GX1 strain (i.e., a strain lacking the Dahms pathway) cultured in medium containing a glucose–xylose mixture. Black symbols indicate the growth of the ATCC31882 (control strain) cultured in medium containing a glucose–xylose mixture. G6P glucose-6-phosphate; PEP phosphoenolpyruvate; E4P erythrose 4-phosphate; and DHS dehydroshikimate. The data are presented as the average of three independent experiments, and error bars indicate standard errors. Source data underlying Fig. 4b are provided as a Source Data file.",
"discussion": "Results and discussion PMP design Our final goal in the present study was to construct an engineered strain that can simultaneously assimilate both glucose and xylose and produce target chemicals with high yields. With this aim, we devised a concept, PMPE. In PMPE, the pathway that produces target chemicals and another pathway that supplies metabolites essential for cell growth and maintenance are completely separated. These two pathways are metabolically parallel in the same strain and do not interfere with each other regarding metabolism (Fig. 1b ). Since there is a larger amount of glucose than xylose in nature, we selected glucose as a substrate for producing target chemicals, and xylose is used in the pathway that synthesizes essential metabolites. Regarding potential parent strains, we chose the previously developed E. coli CFT5 strain 41 . In CFT5, obtained from ATCC 31882, two genes encoding PYR kinase ( pykA and pykF ) were disrupted and the endogenous PTS was replaced with the galactose permease/glucokinase system (GalP/Glk system). These modifications improved PEP availability, and as a result, CFT5 produced various shikimate pathway derivatives at high yields from glucose supplemented in yeast extract. When CFT5 was cultured in M9 minimal medium containing glucose, xylose, or a glucose–xylose mixture, CFT5 did not grow (Supplementary Fig. 2a ). Alternatively, the growth of the CFT5 strain was recovered in medium containing malate, which is a TCA cycle intermediate (Supplementary Fig. 2b ). While the cell growth recovered by adding malate, it needed a very long lag phase, about 2 days. The gene expression patterns of these strains were also evaluated (Supplementary Discussion 1 ). These results suggest that carbon flux from glucose into the TCA cycle is not sufficient to support the growth of the CFT5 strain. The Dahms pathway can produce PYR and glyoxylate, and glyoxylate will be immediately converted to malate via the glyoxylate shunt. Therefore, additional PYR and glyoxylate will be provided by the Dahms pathway. Introducing the Dahms pathway leads to growth on glucose The exogenous xylose catabolic pathway for recovering the cell growth of the CFT5 strain needs to have the following two features: (i) ability to synthesize the essential metabolites of the TCA cycle from xylose and (ii) independence from both glycolysis and PPP. We focused on the Dahms pathway that synthesizes PYR and glyoxylate from xylose without using glycolysis/PPP-related enzymes. In the Dahms pathway, xylose dehydrogenase ( xdh ) from C. crescentus converts xylose to xylonolactone. Xylonolactonase ( xylC ) from C. crescentus converts xylonolactone to xylonate. Xylonate is converted to 2-dehydro-3-deoxy- d -xylonate (DHDOX) by endogenous xylonate dehydratase ( yjhG ), and then DHDOX is converted to PYR and glycolaldehyde by endogenous DHDOX aldolase ( yjhH ) (Supplementary Fig. 1 ). Glycolaldehyde is metabolized to glyoxylate, after which it enters the TCA cycle. A strain, CFT5x, harboring xdh , xylC , and an endogenous yjhHG expression plasmid (Fig. 2a ), was cultured in M9 minimal medium with glucose, xylose, or a glucose–xylose mixture. When xylose-containing medium was used, CFT5x did not grow (Fig. 2b, c ), suggesting that supplying metabolites synthesized from glycolysis or PPP was not sufficient for growth using xylose as the sole carbon source. Unexpectedly, CFT5x grew in M9 minimal medium containing glucose as the sole carbon source, suggesting that introducing the Dahms pathway may activate carbon flow into the TCA cycle from glucose but not xylose. We examined the effect on the cell growth by expressing each enzyme of Dahms pathway, Xdh, CcxylC, and YjhHG in CFT5. As a result, only the strain expressing Xdh could grow in M9 minimal medium containing glucose as the sole carbon source (Supplementary Fig. 3 ). However, Xdh has no or little enzymatic activity for glucose or glucose-6-phosphate as substrates 38 . Thus, 6-phosphogluconate is one of the possible candidates. Fig. 2 Strain construction and cell growth examination. a Metabolic design of CFT5x. b Bacterial cell growth. Blue, black, and red symbols indicate the growth of the CFT5x strain and ATCC31882 cultured in medium supplemented with glucose, a glucose–xylose mixture, and xylose, respectively. c Glucose and xylose consumption. Filled blue symbols indicate glucose consumption in glucose-containing medium. Filled red symbols indicate xylose consumption in xylose-containing medium. Open blue and open red symbols indicate glucose and xylose consumptions in mixed sugar-containing medium, respectively. G6P glucose-6-phosphate; PEP phosphoenolpyruvate; E4P erythrose 4-phosphate; DHS dehydroshikimate; and Gluconate-6P gluconate-6-phosphate. The data are presented as the average of three independent experiments, and error bars indicate standard errors. Source data underlying Fig. 2b, c are provided as a Source Data file. Complete disruption of the metabolic connection One hypothesis of the mechanism of regaining cell growth by expressing Xdh is that Xdh activate the Entner–Doudoroff (ED) pathway. In the ED pathway, 6-phosphogluconate is converted to 2-keto-3-deoxygluconate-6-phosphate by phosphogluconate dehydratase (EDD, EC:4.2.1.12), then it is converted to PYR and glyceraldehyde-3-phosphate by 2-keto-3-deoxygluconate-6-phosphate aldolase, which is encoded by eda . If Xdh has dehydrogenase activity for 6-phosphogluconate as substrate, it is thought that ED pathway would be activated. Therefore, we constructed an eda -deficient strain GXa. Surprisingly, the GXa strain regained cell growth in a minimal medium containing glucose in the absence of the Dahms pathway (Fig. 3 ). This result suggests that the disruption of eda may activate other pathways or provide TCA cycle intermediates. In GXa, ppc and pck which coding PEP carboxylase (Ppc) and PEP carboxykinase (Pck), remained intact. Ppc and Pck catalyze the reaction converting PEP to OAA and the reverse reaction (PEP–OAA interconversion). It is thought that the balance of PEP–OAA interconversion may have been altered by the disruption of the ED pathway. The GXb strain ( ppc disruption) did not grow in M9 minimal medium, but the GXc strain ( pck disruption) regained cell growth in minimal medium. The disruption of ppc resulted in a loss of cell growth (GXb), but the GXf strain (with ppc and pck disruption) could grow. Another pck- disrupted strain, GXe (with eda and pck disruption), exhibited cell growth. Alternatively, the GXd strain (with ppc and eda deletion) exhibited no cell growth (Supplementary Fig. 4a , b ). Fig. 3 Metabolic pathway and growth of CFT5-derived strains. Δ indicates gene disruption. Orange highlighted + indicates that the strain grew in M9 minimal medium with glucose as the sole carbon source. ED pathway Entner–Doudoroff pathway; PEP phosphoenolpyruvate; E4P erythrose 4-phosphate; and 6PG 6-phosphogluconate. In wild-type E. coli , Ppc and Pyk regulate metabolism between glycolysis and the TCA cycle 33 . In the pyk -knockout strain, the activities of Ppc and Pck are higher than those in the wild-type strain 42 . CFT5 also features pykF and pykA disruptions; moreover, it was believed that PEP–OAA interconversion was activated in CFT5-derived strains. Ppc and Pck activity, i.e., PEP–OAA interconversion, depends on the concentration of metabolites in the TCA cycle (i.e., PEP, OAA, acetyl-CoA, and malate), and it is regulated by these metabolites 43 , 44 . Figure 3 suggests that the loss of the growth of the CFT5 and GXb strains was attributable to the regulation of central metabolism including the regulation of Pck by the metabolites rather than insufficient carbon supply. Therefore, we believed that the disruption of these two metabolic pathways (ED pathway and PEP–OAA interconversion) is necessary to completely block carbon flow from glycolysis and PPP into the TCA cycle. In addition, to eliminate unexpected changes in metabolic regulation, all junctions between glycolysis and the TCA cycle need to be disrupted. We generated the GX1 strain (with eda , ppc , pck , and ppsA disruptions). ppsA encodes PEP synthase, which converts PYR to PEP. The GX1 strain did not grow in M9 minimal medium containing glucose. To identify the necessary TCA cycle metabolites for cell growth, the GX1 strain was cultured in PYR-supplemented medium (M9P medium) or PYR- and malate-supplemented media (M9PM medium). It grew in M9PM medium but not M9P medium (Supplementary Fig. 5 ). These results indicate that the GX1 strain has a shortage of citrate, which is the starting material of the TCA cycle, because of the disruption of the oxaloacetate-generating reaction from PEP. In M9PM medium, supplemented malate is converted to oxaloacetate by malate dehydrogenase, which covers the lack of oxaloacetate supply. Growth of the GX1x strain by introducing the Dahms pathway To regain cell growth, we introduced the Dahms pathway into the GX1 strain. GX1x strain equipped with the Dahms pathway (Fig. 4a ) grew in M9 minimal medium with a glucose–xylose mixture as the carbon source, and maximum OD 600 reached at the same level of ATCC31882 (Fig. 4b ). Conversely, the strain did not grow in media containing glucose or xylose alone. Carbon from glucose could not flow into the TCA cycle, which leads to deficiencies of the essential metabolites and energy necessary for cell growth. In the medium containing glucose–xylose mixture, the Dahms pathway supplied PYR and glyoxylate from xylose as carbon sources, thereby restoring cell growth. It was thought that the GX1x strain did not grow in M9 minimal medium containing only xylose because of the insufficient activity of the endogenous xylose catabolic pathway (i.e., xylAB ). We constructed the strain overexpressing XylA and XylB derived from GX1x. Contrary to expectations, this strain did not grow in M9 minimal medium with xylose as a sole carbon source. Furthermore, the strains derived from CFT5 overexpressing Dahms pathway enzymes (Xdh, CcxylC, YjhH, and YjhG) and/or enzymes of the endogenous xylose catabolic pathway (XylA and XylB) also did not grow in the same condition (Supplementary Fig. 6 ). In this strain, while PYR and glyoxylate were supplied via Dahms pathway, these metabolites would have been converted to intermediates of glycolysis and could not be used in the TCA cycle. Additionally, we analyzed the metabolome in PMPE strain. As a result, PMPE strain considerably accumulated PEP and had been enhanced with PPP. These phenotypes are suited for the production of shikimate pathway derivatives (Supplementary Discussion 2 , Supplementary Figs. 14 , 15 ). Fig. 4 Examination of the growth of the strain in which the Dahms pathway was introduced. a Metabolic design of the GX1x strain. b Bacterial cell growth in minimal medium. Gray, blue, and orange symbols indicate the growth of the GX1x strain cultured in medium containing only glucose, a glucose–xylose mixture, and xylose alone, respectively. Yellow symbols indicate the growth of the GX1 strain (i.e., a strain lacking the Dahms pathway) cultured in medium containing a glucose–xylose mixture. Black symbols indicate the growth of the ATCC31882 (control strain) cultured in medium containing a glucose–xylose mixture. G6P glucose-6-phosphate; PEP phosphoenolpyruvate; E4P erythrose 4-phosphate; and DHS dehydroshikimate. The data are presented as the average of three independent experiments, and error bars indicate standard errors. Source data underlying Fig. 4b are provided as a Source Data file. MA production from glucose–xylose mixture in the PMPE strain We attempted to produce MA via the shikimate pathway using a GX1x-derived strain. Protocatechuate (PCA) was synthesized from DHS by DHS dehydratase, PCA decarboxylase converted PCA to catechol (CA), and CA 1,2-dioxygenase produced MA from CA (Supplementary Fig. 7 ) with the highest theoretical yield among all MA synthetic pathways (0.68 g g −1 of glucose) 25 . Figure 5 illustrates the culture profiles of the GX1xMA strain (GX1 harboring an MA-producing plasmid) in M9 minimal medium. The GX1xMA strain produced 1.60 ± 0.08 g L −1 of MA after 96 h of cultivation with a yield of 0.30 ± 0.02 g g −1 of glucose after 72 h. CFT5xMA strain produced 0.71 ± 0.22 g L −1 of MA after 80 h of cultivation with a yield of 0.12 ± 0.05 g g −1 of glucose after 60 h. As a control of glucose and xylose co-utilizing strain, strain CTR2 was constructed by disrupting ptsG and pheA from ATCC31882. These genes are responsible for catabolite repression ( ptsG ) and production of l -phenylalanine competing with MA production ( pheA ), respectively. CTR2MA strain (CTR2 harboring an MA-producing plasmid) was cultured in M9 minimal medium and produced 0.53 ± 0.01 g L −1 of MA after 48 h (Supplementary Fig. 8 ). After 120 h cultivation of CFT5xMA and GX1xMA, 1.63 ± 0.24 and 1.67 ± 0.20 g L −1 of xylonate was accumulated, respectively, and the <0.05 g L −1 of acetate and lactate were accumulated in the medium (Supplementary Fig. 9 ). The production titer of MA from glucose of the GX1xMA strain were 2.3-fold and 3.0-fold higher than CFT5xMA strain and CTR2MA strain, respectively, and the yield was 2.6-fold higher than CFT5xMA. These findings suggest that the complete elimination of carbon flux from glycolysis to the TCA cycle improves MA production. Fig. 5 Culture profiles of cis,cis -muconic acid (MA)-producing strains in M9 minimal medium. a Black, blue, and red symbols indicate bacterial cell growth, glucose consumption, and xylose consumption, respectively. b Green symbols indicate the produced amounts of MA. All open and filled symbols indicate the results for the CFT5xMA and GX1xMA strains, respectively. The data are presented as the average of three independent experiments, and error bars indicate standard errors. P values were computed using the two-tailed Student’s t -test (* P < 0.05). Source data are provided as a Source Data file. 13 C-metabolic analysis of the MA-producing strain To analyze the metabolic flux of glucose and xylose in the PMPE strain, the GX1xMA strain was cultured in M9 minimal medium containing [U- 13 C]glucose and non-labeled xylose. After culturing, the levels of MA in the supernatant and that of five key amino acids (histidine, glycine, alanine, glutamic acid, and lysine) were analyzed via GC–MS. The initial optical density at 600 nm (OD 600 ) of the cultures was 0.5, and cells were harvested for GC–MS analysis when OD 600 reached 2.40 ± 0.47. Figure 6 presents the mass isotopomer distributions of metabolites. MA was completely labeled with 13 C, indicating that all produced MA was derived from glucose. The carbon atoms in histidine were almost completely labeled with 13 C. Histidine is synthesized from the PPP intermediate 5-phosphoribosyl diphosphate. This result indicates that carbon flux into PPP is only derived from glucose, and xylose was not used in this pathway. On the contrary, the proportions of carbon labeled with 13 C in alanine, glutamic acid, and lysine were extremely low. It appears that these amino acids were synthesized from non-labeled xylose. These results support the hypothesis that xylose is mostly catabolized via the Dahms pathway even though the endogenous xylose catabolic pathway (i.e., xylose isomerase and xylulokinase) in E. coli was retained. To prevent accidental activation of the endogenous xylose isomerase pathway, we generated the GX2xMA strain, a GX1xMA-derived strain in which xylA (xylose isomerase) and xylB (xylulokinase) were disrupted. 13 C-metabolic analysis of the GX2xMA strain revealed profiles similar to those of the GX1xMA strain (Fig. 6 ). This result suggested that the metabolites derived from the intermediates of glycolysis or PPP were not sufficiently synthesized from consumed xylose in the strain in which the Dahms pathway was introduced. Fig. 6 13 C-metabolic analysis. Blue, red, and yellow bars indicate the mass isotopomer distributions of six metabolites [glycine, alanine, lysine, histidine, glutamic acid, and cis,cis -muconic acid (MA)] from tracer experiments with [U- 13 C]glucose and non-labeled xylose in ATCC31882xMA, GX1xMA, and GX2xMA, respectively. The vertical axis indicates the relative abundance. The horizontal axis is M+, which denotes the difference with a fully unlabeled isotopomer regarding the m / z of a mass fragment (fully unlabeled isotopomer, M+ = 0). The maximum value of M+ for each metabolite is the number of constituent carbons of the metabolite in the mass fragment. Red closed crosses indicate disrupted metabolic pathways in the GX1 and GX2 strains. Red open cross indicates the disrupted metabolic pathway in the GX2 strain. G6P glucose-6-phosphate; PEP phosphoenolpyruvate; E4P erythrose 4-phosphate; and αKG alpha-ketoglutarate. The data are presented as the average of three independent experiments, and error bars indicate standard errors. Source data are provided as a Source Data file. Contrary to our expectations, both labeled and non-labeled carbon atoms were detected in glycine and alanine. This was caused by the interconversion of serine and PYR 45 , as catalyzed by serine deaminase (SDA) encoded by sdaA . The interconversion of 13 C-labeled serine and non-labeled PYR appears to be the junction between the glucose-dominated glycolysis/PPP and xylose-dominated TCA cycle. We generated the GX3xMA strain, which was derived by disrupting sdaA in the GX1xMA strain. The GX3xMA strain could not grow in M9 minimal medium containing the sugar mixture. These results indicate that although carbon metabolism mediated by SDA is essential for the growth of GX1-derived strain in M9 minimal medium, the ratio of carbon leakage was small in relation to the entire metabolism of E. coli . Optimization of MA production using PMPE strain For MA production, the optimal glucose:xylose ratio was determined using the GX1xMA strain, which was cultivated using M9 minimal medium containing 20 g L −1 sugar(s) and 5 g L −1 yeast extract (M9Y medium) in shake flasks. The ratio of glucose to total sugars was 0%, 25%, 50%, 75%, or 100%. As expected, the cells did not grow well in medium containing glucose or xylose alone (Fig. 7a ), corresponding to the results presented in Fig. 4b . Both glucose and xylose were needed for cell growth, and glucose content exceeding 50% was required. The highest specific growth rate was 0.136 h −1 in 75% glucose-containing medium (Supplementary Table 1 ). Fig. 7 Culture profiles of the GX1xMA strain in M9 minimal medium containing 20 g L −1 sugar(s) and 5 g L −1 yeast extract. a Bacterial cell growth. b Produced amounts of MA. c Glucose consumption. d Xylose consumption. e Production yield of cis,cis -muconic acid (MA) from glucose (g of produced MA g −1 of consumed glucose). Red, orange, yellow, purple, and blue symbols indicate the results of cultivation in 0%, 25%, 50%, 75%, and 100% glucose medium, respectively, in a – h . f Culture profiles of the GX1xMA strain with the addition of CaCO 3 . The final sampling time is after 80 h cultivation. Blue, red, and yellow symbols indicate the glucose concentration, xylose concentration, and the produced amount of MA, respectively. The data are presented as the average of three independent experiments, and error bars indicate standard errors. Source data are provided as a Source Data file. Figure 7b presents the results of MA production. The GX1xMA strain produced 0.84, 1.94, and 3.13 g L −1 of MA after 48 h of cultivation in 25%, 50%, and 75% glucose-containing media, respectively. MA yields were similar among these glucose ratios (Fig. 7e ). When using [U- 13 C]glucose and non-labeled xylose as substrates, the produced MA was completely labeled with 13 C (Supplementary Fig. 10 ). Although the titer was increased by optimizing the glucose:xylose ratio, the yield decreased compared with that presented in Fig. 5 (titer, 1.60 ± 0.08 g L −1 ; yield, 0.30 ± 0.02 g g −1 of glucose). The maximum productivity of MA in 25%, 50%, and 75% glucose-containing media were 0.078, 0.072, and 0.114 g L −1 h −1 , respectively. The intermediates of the MA synthesis pathway (PCA and CA) and shikimate pathway derivatives ( l -phenylalanine, l -tyrosine, l -tryptophan, p -aminobenzoate, and p -hydroxybenzoate) were not detected in the medium, suggesting that these pathways were not rate-limiting steps. We found that several organic acids were accumulated as by-products. As the xylose ratio in the medium was increased, considerable acetate and lactate productions were noted. In 25% glucose medium (glucose:xylose ratio is 1:3), 1.87 g L −1 of acetate and 1.29 g L −1 of lactate were produced after 48 h of cultivation (Supplementary Fig. 11 ). It is considered that the excessively consumed xylose overflowed to these organic acids. It is thought that the accumulation of acetate was caused by the activation of phosphate acetyltransferase (Pta) due to the accumulation of PEP. Pta converts PYR to acetyl phosphate and acetyl phosphate is converted to acetate by acetate kinase. PEP acts as a regulator of Pta and activates the production of acetyl phosphate and inhibit the reverse reaction 46 . The accumulation of lactate might be caused by a decrease in pH. In all conditions, pH was decreased under 5.0 after 72 h cultivation. The low pH condition activates lactate dehydrogenase which converts PYR to lactate 47 . Furthermore, the accumulation of xylonate and glyoxylate increased in proportion to xylose concentration (Supplementary Fig. 11 ). In the case of medium containing xylose as the sole carbon source, 11.11 g L −1 of xylonate was produced after 72 h of cultivation. Xylonate accumulation is a common problem of Dahms pathway-mediated chemical production 39 , 40 , 48 , 49 , which decreases the yields of target compounds synthesized via this pathway. To reduce the accumulation of xylonate, overexpressing lactaldehyde dehydrogenase (AldA) would be one of the possible strategies. Glycolaldehyde synthesized via Dahms pathway is converted to glycolate by AldA and then glycolate is converted to glyoxylate by glycolate oxidase. Cabulong et al. increased glycolic acid production by overexpressing AldA 39 . In PMPE strains, it might be possible to improve the accumulation of xylonate that pulling the carbon flow from glycolaldehyde to glyoxylate by overexpressing AldA and glycolate oxidase. Another explanation of the glyoxylate accumulation is due to the shortage of acetyl-CoA. Glyoxylate and acetyl-CoA are converted to malate by malate synthase. Disruption of Pta and overexpression of malic enzymes coded by maeB (NADP-dependent malic enzyme) or sfcA (NAD-dependent malic enzyme), which converts malate to PYR, have the potential to solve glyoxylate accumulation. Furthermore, organic acids accumulation reduced the pH of the medium (4.59, 4.46, 4.85, and 4.98 in 0%, 25%, 50%, and 75% of glucose medium after 72 h of cultivation, respectively). Because pH control is important for MA production 29 , CaCO 3 (10 g L −1 ) was added to the culture medium after 24 h of cultivation (Fig. 7f ). Under this condition, the GX1xMA strain produced 4.09 ± 0.14 g L −1 of MA after 72 h of cultivation (Fig. 7f ), representing a 1.31-fold increase compared with that produced without CaCO 3 . The yield reached 0.31 ± 0.003 g g −1 of glucose (80 h), which was improved to the same level as that in M9 minimal medium (Fig. 5 ). It is believed that carbons from glucose flowed to more downstream metabolites or that biomass turnover occurred via the degradation of shikimate pathway derivatives. To further increase the production and yield, it is necessary to disrupt metabolic pathways that compete with the shikimate pathway, and aroE and ydiB , which encode shikimate dehydrogenase, are promising candidates for such disruption 25 , 50 (Supplementary Fig. 7 ). We demonstrated MA production using the PMPE strain. Figures 5 and 7f show that CCR was almost relaxed, and glucose and xylose consumptions were simultaneously achieved. In many studies, xylose was catabolized via XylAB after the deregulation of CCR. Generally, xylose-metabolizing pathways catalyzed by XylAB have lower activity than glucose catabolism through glycolysis, as shown in Fig. 2 . Our PMPE strain featuring the Dahms pathway consumed and efficiently metabolized xylose, leading to cell growth and high MA production. Moreover, the PMPE strategy simplifies the process compared with co-culture systems and the operation of fermentation using glucose–xylose mixtures. Production of other target metabolites using PMPE strain To confirm the versatility of PMPE in the shikimate pathway, we attempted to produce l -tyrosine using the PMPE strain. The GX1xTYR strain, a GX1-derived strain harboring tyrA fbr encoding feedback-resistant chorismate mutase/prephenate dehydrogenase, was cultivated in M9Y medium containing both 3.75 g L −1 of glucose and 1.25 g L −1 of xylose in shake flasks. In aerobic condition, GX1xTYR strain produced 1.34 ± 0.05 g L −1 of l -tyrosine after 96 h of cultivation, representing a 1.73-fold increase compared with that produced by the control strain CFT5xTYR (Fig. 8 ). The maximum productivity of l -tyrosine in CFT5xTYR and GX1xTYR were 0.026 and 0.021 g L −1 h −1 , respectively. Xylonate, glyoxylate, lactate, and acetate were not accumulated in the medium after the cultivation of CFT5xTYR or GX1xTYR. The production yield reached 0.35 ± 0.01 g g −1 of glucose after 96 h of cultivation, reflecting a 2.15-fold increase compared with that using the CFT5xTYR strain and corresponding to 64% of the theoretical maximum without considering cell growth (0.55 g g −1 of glucose) 51 . In micro-aerobic condition, GX1xTYR strain and CFT5xTYR strain produced 0.32 ± 0.02 g L −1 (96 h) and 0.40 ± 0.02 g L −1 (72 h) of l -tyrosine, respectively, and the production yield in GX1xTYR reached 0.32 ± 0.10 g g −1 of glucose after 96 h of cultivation, reflecting a 5.22-fold increase compared with that using the CFT5xTYR (Fig. 8 ). These results suggested that PMPE is superior also in micro-aerobic conditions at the production yield, however, the production titer needs to be improved. In micro-aerobic condition, CFT5xTYR and GX1xTYR produced by-products (such as organic acids and ethanol) and the expression level of aerobic respiration control protein (ArcA, coded by arcA ) was increased in micro-aerobic conditions (Supplementary Fig. 12a , b ). ArcA down-regulates the transcription of genes in the TCA cycle 52 . To improve the productivity of PMPE strain in micro-aerobic conditions, the engineering of metabolic regulators including ArcA would be necessary. Juminaga et al. improved l -tyrosine production by deregulating bottlenecks (AroB and AroK) in its biosynthetic pathway 53 . They identified the bottlenecks by considering the expression way of genes. The optimized strain produced 2.17 g L −1 of l -tyrosine, which corresponds to 80% of the theoretical yield. Although the expression of shikimate pathway enzymes was not optimized in the PMPE strain, the achieved yield of l -tyrosine was high. It is believed that blocking carbon flow into the TCA cycle, which competes with the shikimate pathway, improves the yield of l -tyrosine production without optimizing enzyme expression in the shikimate pathway. To further improve l -tyrosine production, modifying the expression of shikimate pathway enzymes in the PMPE strain is a promising strategy. In our previous study, we suggested that a major reason for decreased yields of shikimate pathway derivatives was the loss of carbon for synthesizing metabolites branching from chorismate, which is an end-product of the shikimate pathway 29 . We solved this problem by expressing the protein fused chorismate synthase and isochorismate synthase in MA-producing strain in this study. This technique may be applicable to the PMPE strain for further improving l -tyrosine production. Additionally, we constructed the strain producing 1,2-propanediol as a non-shikimate pathway chemical derived from GX1x (GX1xPD). GX1xPD increased the production titer and the yield of 1,2-propanediol by 1.40-fold and 11.4-fold, respectively, compared to the control strain. (Supplementary Discussion 3 , Supplementary Figs. 16 , 17 ). These results suggested that PMPE is versatile for applying the production of various compounds including non-shikimate pathway derivatives. Fig. 8 Cultures profiles of l -tyrosine-producing strains. a , b Aerobic condition. c , d Micro-aerobic condition. a , c Black, blue, and red symbols indicate bacterial cell growth, glucose consumption, and xylose consumption, respectively. b , d Green symbols indicate the produced amounts of l -tyrosine. All open symbols and filled symbols indicate the results for the CFT5xTYR and GX1xTYR strains, respectively. The data are presented as the average of three independent experiments, and error bars indicate standard errors. P values were computed using the two-tailed Student’s t -test (* P < 0.05; ** P < 0.01). Source data are provided as a Source Data file. We demonstrated that PMPE enables the efficient use of glucose–xylose co-substrate, and this strategy can be applied for producing shikimate pathway derivatives. The loss of cell growth caused by the complete interruption of carbon flow into the TCA cycle could be rescued by introducing the Dahms pathway. A high yield of MA was achieved using the PMPE E. coli strain because all glucose was used for target chemical production, whereas essential metabolites were supplied from xylose via the Dahms pathway. Poor growth and xylonate accumulation should be improved in the PMPE E. coli strain. Xylonate dehydratase in the Dahms pathway is a rate-limiting enzyme, and another isozyme, such as E. coli yagF with higher enzyme activity 39 would be useful for efficient utilization of xylose. Glyoxylate accumulation is another issue to be solved. Using Weimberg pathway is an alternative option because this pathway supplies α-ketoglutarate from xylose without producing glyoxylate 54 . By changing the disconnection point of metabolism, PMPE would be applicable to other pathways that branch from PYR, acetyl-CoA, and the upstream metabolites of glycolysis. Chemicals such as 1,2-propanediol are produced under anaerobic conditions 55 , 56 to repress carbon flow into the TCA cycle, which causes a shortage of NADH supply 57 . We also demonstrated that PMPE can be applied to the production of 1,2-propanediol. Therefore, it is expected that the PMPE strain will significantly contribute to the production of other non-shikimate pathway chemicals with high yields."
} | 11,444 |
24110243 | null | s2 | 4,243 | {
"abstract": "Emerging multi-electrode-based brain-machine interfaces (BMIs) and large multi-electrode arrays used in in vitro experiments, enable recording of single neuron's activity on multiple electrodes and allow for an in-depth investigation of neural preparations, even at a sub-cellular level. However, the use of these devices entails stringent area and power consumption constraints for the signal-processing hardware units. In addition, the high autonomy of these units and an ability to automatically adapt to changes in the recorded neural preparations is required. Implementing spike detection in close proximity to recording electrodes offers the advantage of reducing the transmission data bandwidth. By eliminating the need of transmitting the full, redundant recordings of neural activity and by transmitting only the spike waveforms or spike times, significant power savings can be achieved in the majority of cases. Here, we present a low-complexity, unsupervised, adaptable, real-time spike-detection method targeting multi-electrode recording devices and compare this method to other spike-detection methods with regard to complexity and performance."
} | 289 |
20166230 | null | s2 | 4,244 | {
"abstract": "Silk fibroin is a useful protein polymer for biomaterials and tissue engineering. In this work, porogen leached scaffolds prepared from aqueous and HFIP silk solutions were reinforced through the addition of silk particles. This led to about 40 times increase in the specific compressive modulus and the yield strength of HFIP-based scaffolds. This increase in mechanical properties resulted from the high interfacial cohesion between the silk matrix and the reinforcing silk particles, due to partial solubility of the silk particles in HFIP. The porosity of scaffolds was reduced from approximately 90% (control) to approximately 75% for the HFIP systems containing 200% particle reinforcement, while maintaining pore interconnectivity. The presence of the particles slowed the enzymatic degradation of silk scaffolds."
} | 205 |
37215776 | PMC10199210 | pmc | 4,245 | {
"abstract": "Microbial electrochemical technologies (METs) are a group of innovative technologies that produce valuables like bioelectricity and biofuels with the simultaneous treatment of wastewater from microorganisms known as electroactive microorganisms. The electroactive microorganisms are capable of transferring electrons to the anode of a MET through various metabolic pathways such as direct (via cytochrome or pili) or indirect (through transporters) transfer. Though this technology is promising, the inferior yield of valuables and the high cost of reactor fabrication are presently impeding the large-scale application of this technology. Therefore, to overcome these major bottlenecks, a lot of research has been dedicated to the application of bacterial signalling, for instance, quorum sensing (QS) and quorum quenching (QQ) mechanisms in METs to improve its efficacy in order to achieve a higher power density and to make it more cost-effective. The QS circuit in bacteria produces auto-inducer signal molecules, which enhances the biofilm-forming ability and regulates the bacterial attachment on the electrode of METs. On the other hand, the QQ circuit can effectively function as an antifouling agent for the membranes used in METs and microbial membrane bioreactors, which is imperative for their stable long-term operation. This state-of-the-art review thus distinctly describes in detail the interaction between the QQ and QS systems in bacteria employed in METs to generate value-added by-products, antifouling strategies, and the recent applications of the signalling mechanisms in METs to improve their yield. Further, the article also throws some light on the recent advancements and the challenges faced while incorporating QS and QQ mechanisms in various types of METs. Thus, this review article will help budding researchers in upscaling METs with the integration of the QS signalling mechanism in METs.",
"conclusion": "6 Conclusion The METs are an innovative set of technologies for concomitant wastewater treatment with valuable resource recovery. However, high fabrication cost and lower yield is the major obstacle to the way of commercialization of the METs. In this regard, QSM and QQM secreted by different bacterial strains can be employed to improve the yield of valuables through METs. The QSMs enhance the interaction of electron-carriers within the anodic chamber in METs and the QQMs, in turn, salvage the detrimental effects of the thick bacterial biofilms on the electrodes. Moreover, because of their networking nature, QSM is proven as an efficient tool for the development of firm biofilm, which results in the improvement in valuables recovered through METs. Furthermore, QQM has also gained considerable attention among the scientific community for its property of preventing the biofouling of membranes in METs without causing any disturbance to the beneficial microbes. Hence, the QS-QQ signalling mechanism is a viable approach for enhancing the efficacy of METs in terms of valuable resource recovery with concomitant wastewater treatment.",
"introduction": "1 Introduction Microbial electrochemical technologies (METs) are promising techniques for wastewater treatment with concomitant value-added product recovery. The METs utilize microorganisms for catalysing different electrochemical reactions and thus can be characterized as microbial fuel cell (MFC) for bioelectricity production, microbial electrolysis cell (MEC) for hydrogen production, microbial electrosynthesis (MES) used for chemical recovery, microbial desalination cell for desalinization of saline water and microbial carbon capture for carbon sequestration with bioenergy recovery [ 1 ]. The METs are unique and clean technologies which are a combination of electrochemical and biological methods for wastewater treatment and concomitant energy recovery in terms of bioelectricity and valuable products such as; hydrogen, acetate, hydrogen peroxide and methane etc [ 2 ]. In METs, the organic compounds present in the wastewater act as the substrate for the anaerobic microbes present in the anodic chamber; on the other hand, in the cathodic chamber, oxygen acts as the terminal electron acceptor in the oxygen reduction reaction. The working mechanism of METs is purely dependent on the electroactive microorganisms formally termed as biocatalysts, which oxidizes the organic fraction of wastewater and generates electrons and protons. These produced electrons and protons are then further utilized based on the type of MET reactor used, such as in MFC; these protons and electrons are transferred through a proton exchange membrane (PEM) and an external circuit, respectively, to the cathode, where the oxygen reduction reaction takes place leading to the production of energy in the form bioelectricity. Similarly, for microbial carbon capture, oxygen required for the cathodic reaction is provided by the algal cells cultured in this chamber, thus reducing the dependence on external aeration. Whereas in the case of MEC, exoelectrogens oxidize the organic matter present in wastewater in the anodic chamber and produce CO 2 , protons, and electrons [ 3 ]. Further, the electrons move to the cathodic chamber via an external circuit and the proton passes through the PEM to the cathodic chamber and simultaneously gets reduced to form H 2 ( Fig. 1 ). Fig. 1 Schematic of a microbial fuel cell. Fig. 1 In recent times, METs have gained significant attention due to their promising applications for valuables recoveries such as bioelectricity, biohydrogen, biomethane, commodity chemicals and biofuels. The METs are greener technologies for resource recovery with concomitant wastewater treatment without the emission of any greenhouse gases and less sludge generation. However, the inferior yield of resources and higher fabrication cost are the major setbacks for the METs, which limits these technologies to lab-scale only. The word “quorum sensing” refers to sensing the environment by bacteria for an appropriate physiological response and gene expression during transcription under a certain threshold cell density [ 4 ]. This is operated by signal molecules known as auto-inducers (AI) produced by the bacteria and exchanged with the surrounding environment, when the cell density of autoinducers is high [ 5 ]. The production of AIs depends upon the bacterial cell density and is produced in direct proportion to the number of bacteria present in the colony [ 6 ]. Numerous signal molecules, known as QS molecules (QSM), can be found within a bacterial population and these are primarily categorized into four groups depending on the nature of AIs, namely, i) Acyl-homoserine lactones (AHLs); ii) oligopeptides or autoinducing peptides (AIP) iii) autoinducer-2 (AI-2) iv) Pseudomonas quinolone signal (PQS) and autoinducer-3 (AI-3). The mechanism of quorum sensing (QS), which is dependent upon the expression of genes, is controlled by transcription and is common in homogeneous or heterogenous bacterial communities [ 7 , 8 ]. The QSMs contribute to a variety of physiological changes, like pathogenicity, complex formation, symbiosis, competence, antibiotic synthesis, as well as the formation of biofilm. Moreover, the bacterial QS mechanism is cellular communication between bacteria in response to extracellularly secreted AIs. Fundamental processes necessary for the survival of bacterial colonies viz; biofilm formation, antibiotic quorum quenching (QQ) production, sporulating capacity, bioluminescence, activation of virulence genes, and competence regulation are controlled by the QS mechanism [ 9 , 10 ]. The AIs are signalling molecules which are produced intracellularly and secreted by the cells either by passive or active transport. Usually, AIs are present outside the cells at a concentration low enough to be detected by other bacterial cells [ 10 ]. At low cell densities of microbial cells, the AIs are low in number; however, at high cell densities, there are enough AIs for the detection and regulation of gene expression programs [ 11 ]. In this regard, the application of QS and QQ for improving the performance of METs has gained considerable attention because of their ability to enhance the yield of valuables obtained through METs. Basically, the biofilm formation because of the attached growth on the electrode improves the performance of METs by reducing the losses associated with electron transfer. However, excessive profuse biofilm formation on the surface of the electrode and PEM deteriorates the performance of METs [ 12 ]. Therefore, the performance of MFC is dependent on the thickness of the biofilm; moreover, biofouling of membranes also gets triggered due to the random bacterial growth on the membrane surface, which diminishes the performance of membranes and results in the poor performance of the METs as well [ 13 ]. The optimal biofilm thickness on the surface of the anode and prevention of membrane biofouling can be achieved by interfering with the QS communication, thus improving the overall efficacy of the METs. Therefore, the inhibition of the autoinducer-mediated QS mechanism known as QQ can control the anodic biofilm as well as membrane biofouling [ 14 ]. Hence, due to the controlled biofilm formation, the electron transfer becomes efficient, which ultimately enhances the performance of METs. The mechanism of QQ on the other hand, is a mechanism of inhibiting the QSMs through the production of various QQ molecules (QQM). The QQMs are found in nature (i.e., bacteria, fungi, plants), like chemical AI analogues and enzymes isolated from microorganisms. Hence, bacteria can themselves monitor their population growth by bringing variations in the concentration of signal molecules for upregulation or downregulation of a particular gene and the generated signals regulate the process of QS & QQ [ 15 ]. The QQMs also inhibit biofilm-formation by regulating the expression of gene clusters (operons) in bacteria. The bacteria form biofilms as a part of social behaviour to interact with each other via the QS-QQ signalling mechanisms and this aids in the exchange of nutrients and gases, electrons, and protection from pathogenic attacks [ 12 ]. In wastewater treatment through METs, QQ and QS plays an important role as antifouling agent to prevent the pathogenicity of biofilms, improving the longevity of the membrane [ 16 ]. The QSMs control cell-cell communication and aid in electron exchange in METs via biofilm formation, whereas the QQMs regulate the thickness of biofilms to optimise effluent treatment and value-added product recovery [ 17 ]. Bacterial biofilm formation in the anodic chamber of the MFC increases the rate of degradation of organics and electron exchange, which is advantageous for METs [ 18 ]. However, thick anodic biofilms reduce electron transportation as the bacteria in the inner layers do not have as much access to the substrate compared to the ones on the outer surface, accompanied by the loss of substrate from the outer to inner layers of the biofilm [ 19 ]. It has also been observed that electron transportation is less efficient in the inner biofilm layers [ 14 ]. Thick biofilm formation on the membrane, formally termed as membrane biofouling, also affects the proton exchange via the ion exchange membrane used in METs, thus, interfering with bioelectricity production [ 14 ]. Therefore, anodic biofilms with optimal thickness are prerequisites for the efficacious performance of METs. Several biological or pharmacological approaches, including QQ and enzymatic processes, have proved to be effective for membrane cleaning and preventing membrane biofouling [ 20 ]. Besides, silver nanoparticles and poly-sulphones have been able to mitigate biofouling in METs [ 21 ]. It is widely known that the AHL-intervened QS plays a significant role in the formation of biofilm. As a result, numerous techniques for AHL degradation have been developed to block this QS and biofouling is mitigated [ 22 ]. Biocides and other antifouling agents have also been employed in the recent past to tackle biofouling in METs. Das et al. (2021) have shown that CuMnFe composite in a 25 L pilot-scale MFC can be successfully used as an antifouling agent [ 23 ]."
} | 3,060 |
21966078 | null | s2 | 4,246 | {
"abstract": "Experiments have shown that wild type P. aeruginosa swarms much faster than rhlAB mutants on 0.4% agar concentration surface. These observations imply that development of a liquid thin film is an important component of the self-organized swarming process. A multiscale model is presented in this paper for studying interplay of key hydrodynamical and biological mechanisms involved in the swarming process of P. aeruginosa. This model combines a liquid thin film equation, convection-reaction-diffusion equations and a cell-based stochastic discrete model. Simulations demonstrate how self-organized swarming process based on the microscopic individual bacterial behavior results in complicated fractal type patterns at macroscopic level. It is also shown that quorum sensing mechanism causing rhamnolipid synthesis and resulting liquid extraction from the substrate lead to the fast swarm expansion. Simulations also demonstrate formation of fingers (tendrils) at the edge of a swarm which have been earlier observed in experiments."
} | 258 |
29358378 | PMC5819412 | pmc | 4,248 | {
"abstract": "Significance Replacement of nonrenewable petrochemicals and liquid fuels requires sustainable production of oleochemicals. Free fatty acids (FFAs) are versatile molecules that can be produced by microbial fermentation and are used as precursors for production of these oleochemicals. In the past few years, we have seen major advancements in improving the yeast Saccharomyces cerevisiae for FFA production. Despite these successes, lipid metabolism is highly complex, and the pathways and metabolites involved in the formation of FFAs in yeast remain incompletely understood. In this work, we make important advancements in understanding the dynamics of FFA formation in the cell and explore the role of phospholipids in this process.",
"discussion": "Discussion Regulating levels of different lipid pools is an evolutionary advantage for a yeast cell, providing the capacity to adapt to different environments or carbon source availability and resistance to stresses. However, as a chassis for cell factory development and metabolic engineering, a complex metabolic network entwined with tight regulation mechanisms can offer many challenges and difficulties for progress. Here we have engineered the lipid metabolism of S. cerevisiae by removing many reactions involved in the production of storage lipids, fatty acid oxidation, and conversion of FFAs to fatty acyl-CoA. The combined deletions not only were relevant in reducing the complexity of the metabolic network, but also disrupted several lipid regulation mechanisms. Strains resulting from constraining the lipid metabolic network and redirecting fatty acid fluxes toward phospholipids showed increases in both FFAs and phospholipids, suggesting a strong correlation between the two. Characterization of the resulting strain MLM1.0 points strongly toward phospholipid hydrolysis as a major pathway for FFA formation, identifying phospholipases B as the key players in this process. The process of deregulating fatty acid and phospholipid biosynthesis genes led to significant changes in the distribution and total levels of most analyzed lipid classes. It also caused severe morphological changes in the internal membrane structure. Taken together, our results highlight the complexity of entwined metabolic and regulatory networks found in S. cerevisiae lipid metabolism. By identifying the different factors that come into play and how the lipid species are redistributed in the cell on the redirection of lipid metabolism, we believe to have contributed important information toward understanding the effects that emerge when S. cerevisiae is engineered at the level of lipid metabolism. While the strains developed in this study strongly contribute to fundamental insights into FFAs dynamics, they can also serve as a tool for the development of S. cerevisiae as a cell factory for the production of FFAs and fatty-acid derived products. One of the favorable traits of S. cerevisiae is its capacity to accumulate high levels of precursors without the tight feedback regulation mechanisms present in lipid metabolism and the lack of major competing reactions for acyl-CoA. Furthermore, the strain was developed through scarless deletions, without the use of resistance markers or other common genetic elements. This offers major advantages for further studies, because it does not limit genome editing options. Along with the advantages of reduced complexity of the lipid metabolic network with improved regulation, the strain also has the benefit of not carrying any overexpression, which means that the strain is not suffering from a protein burden. On the contrary, through the deletion of several genes, it may be possible to allocate proteome mass for the expression of heterologous pathways that further convert the FFAs to other valuable products, such as fatty alcohols, olefins, or alkanes."
} | 964 |
35280713 | PMC8894036 | pmc | 4,250 | {
"abstract": "The task of designing an Artificial Neural Network (ANN) can be thought of as an optimization problem that involves many parameters whose optimal value needs to be computed in order to improve the classification accuracy of an ANN. Two of the major parameters that need to be determined during the design of an ANN are weights and biases. Various gradient-based optimization algorithms have been proposed by researchers in the past to generate an optimal set of weights and biases. However, due to the tendency of gradient-based algorithms to get trapped in local minima, researchers have started exploring metaheuristic algorithms as an alternative to the conventional techniques. In this paper, we propose the GGA-MLP (Greedy Genetic Algorithm-Multilayer Perceptron) approach, a learning algorithm, to generate an optimal set of weights and biases in multilayer perceptron (MLP) using a greedy genetic algorithm. The proposed approach increases the performance of the traditional genetic algorithm (GA) by using a greedy approach to generate the initial population as well as to perform crossover and mutation. To evaluate the performance of GGA-MLP in classifying nonlinear input patterns, we perform experiments on datasets of varying complexities taken from the University of California, Irvine (UCI) repository. The experimental results of GGA-MLP are compared with the existing state-of-the-art techniques in terms of classification accuracy. The results show that the performance of GGA-MLP is better than or comparable to the existing state-of-the-art techniques.",
"conclusion": "6. Conclusion and Future Work In this paper, a greedy genetic algorithm, GGA-MLP, is presented to train MLP. The use of domain-specific knowledge enables the generation of good quality initial population. Mean-based crossover and greedy mutation help algorithm in moving toward global optima by exploring the search space thoroughly. Datasets of varying complexities are used to evaluate the performance of GGA-MLP and to compare it with existing state-of-the-art algorithms as well as existing classifiers such as Naïve Bayes, decision tree, logistic regression, and MLP trained using BP. The results show that although GGA-MLP takes more time to converge as compared to other metaheuristic algorithms, the performance of GGA-MLP is better than or comparable to the existing techniques in classifying datasets, especially large datasets, as GGA-MLP searches the solution space properly by maintaining a balance between exploration and exploitation. In future, we plan to extend our work to train other types of ANNs and incorporate architecture optimization in it.",
"introduction": "1. Introduction Artificial Neural networks (ANNs) are computing models inspired by the biological nervous system. An ANN consists of an interconnected network of nodes called artificial neurons which are organized in the form of layers, namely, input layer, hidden layers, and output layer [ 1 ]. A set of synaptic weights is used to interconnect the nodes that form these layers. ANNs have been applied to a broad range of problems like classification, regression, prediction, pattern recognition, and disease diagnosis [ 2 – 6 ]. Classification is one of the important areas of research in the field of data science. Many classification models exist, out of which ANNs are among the most widely used models. In this paper, our focus is on multilayer perceptron (MLP) which is a multilayer feedforward neural network. Classification using MLP is basically a two-step process. The first step is the learning (training) phase in which a classifier is built to describe a predetermined set of data classes for a given dataset (training data). In the second step, the model which has been built in the training phase is used for the classification of the unclassified data (test data) for estimating the accuracy of the classifier. During the learning phase, MLP learns by adjusting synaptic weights and biases iteratively in an attempt to correctly predict the class labels of the input data. The process of weight and bias update continues until the acquired knowledge is sufficient and the network reaches a specified level of accuracy; i.e., a predefined error measure is minimized, or the maximum number of epochs is reached [ 7 ]. After the completion of the learning phase, it is mandatory to assess the performance of MLP, i.e., its generalization and predictive capabilities, using samples of data (test data) that are different from those used during the training phase for the given dataset. To achieve generalization, MLPs need to avoid the issues of both underfitting and overfitting during the training phase. To achieve the best results, it is therefore required that the number of training patterns should be sufficiently larger than the total number of connections in the neural network. The performance of MLP is highly dependent on the learning method used to train it during the training phase. Several learning algorithms exist in the literature with the aim of finding an optimal MLP. These learning algorithms can be broadly classified into three categories, namely, conventional methods [ 8 – 12 ], metaheuristic-based methods [ 13 – 37 ], and hybrid methods [ 20 , 38 – 44 ]. Despite the existence of a large number of learning algorithms, researchers continue to apply new optimization techniques like multimean particle swarm optimization (MMPSO) [ 28 ], whale optimization algorithm (WOA) [ 23 ], multiverse optimizer (MVO) [ 34 ], grasshopper optimization algorithm (GOA) [ 35 ], and firefly algorithm [ 36 ] to generate an optimal set of synaptic weights in an attempt to further improve the accuracy and performance of MLP. As stated in No-Free-Lunch (NFL) theorem [ 45 ], there is no optimization technique that solves all optimization problems. It is quite possible that an existing learning algorithm may train an MLP well for some datasets while it fails to do the same for some other datasets. This makes the field of generating optimal connection weights a dynamic research area. This is the main motivation behind the work presented in this paper, in which we propose a hybrid learning algorithm to train MLP. GA is an evolutionary algorithm (EA) and is one of the most widely investigated algorithms among the metaheuristic algorithms in designing neural networks. Over the years, GA and its variants have been successfully applied in several domains for ANN weight [ 13 – 20 ], topology [ 46 – 48 ], and feature set optimization [ 49 , 50 ], as well as parameter tuning [ 51 , 52 ]. A comprehensive review of optimization of neural networks using GA can be found in [ 53 ]. The efficiency, effectiveness, and ease of use of GA motivated us to further improve the performance of GA in optimizing weights of MLP by integrating greedy techniques with GA. The proposed algorithm Greedy Genetic Algorithm–Multilayer Perceptron (GGA-MLP) improves the performance of traditional GA by using a greedy approach to generate the initial population as well as to perform crossover and mutation. Some of the application areas of the proposed work are disease identification, e-mail spam identification, prediction of the stock market, and fruit classification. The main challenge with the proposed approach is that it may not work well with some of the datasets, as stated by No-Free-Lunch (NFL) theorem [ 45 ] mentioned above. Finally, the performance of GGA-MLP is compared with various classifiers as well as the existing state-of-the-art metaheuristic algorithms for training MLP. The key contributions of this paper are as follows: A hybrid learning algorithm, GGA-MLP, that integrates greedy techniques with GA is proposed to train MLP GGA-MLP is evaluated and compared with existing state-of-the-art algorithms on 10 datasets of different complexities The paper is organized as follows. Related work is presented in Section 2 . A brief overview of GA is given in Section 3 . In Section 4 , the proposed GGA-MLP for optimization of MLP weights and biases is presented. In Section 5 , experiments conducted to evaluate the effectiveness of GGA-MLP are presented, and results are discussed. Finally, the conclusion and future work are discussed in Section 6 .",
"discussion": "5. Results and Discussion First, we present the datasets that are selected to evaluate the effectiveness of GGA-MLP, in terms of accuracy achieved in classifying the input data, in Section 5.1 . The implementation details, experimental setup used for performing experiments, and results are presented in Section 5.2 . 5.1. Datasets To evaluate the effectiveness of the proposed approach GGA-MLP, ten standard binary classification datasets are selected from the UCI Machine Learning Repository [ 61 ]: Parkinson, Indian Liver Patient Dataset (ILPD), Diabetes, Vertebral Column, Spambase, QSAR Biodegradation, Blood Transfusion, HTRU2, Drug Consumption: Amyl Nitrite, and Drug Consumption: Ketamine. The description of the selected datasets is shown in Table 1 . In each dataset, 80% of the instances are used for training (out of which 20% is used for validation), and the remaining 20% are used for testing. It can easily be seen from Table 1 that the selected datasets have different numbers of features ranging from 4 to 57 as well as instances ranging from 197 to 17898, which helps us to evaluate the proposed approach on datasets of varying complexities. It also makes the task of evaluating GGA-MLP even more challenging. 5.2. Experimental Design and Results To evaluate the effectiveness of GGA-MLP, the performance of MLP trained using GGA-MLP is compared with the classification accuracy of MLP trained using existing algorithms, namely, GA [ 21 ], ABC [ 27 ], MMPSO [ 28 ], WOA [ 33 ], MVO [ 34 ], and GOA [ 35 ], and on each dataset given in Table 1 . All the algorithms are implemented in Python 3.6.4 using the Anaconda framework. As these are randomized algorithms, 30 runs of each algorithm are performed on every dataset. After each run, the best MLP is selected, and its classification accuracy on the test dataset is calculated using (5) C l a s s i f i c a t i o n A c c u r a c y = N C N , where NC is the number of correctly classified testing data samples and N is the total number of samples in the testing dataset. Before the start of the training phase, it is required to decide the architecture of MLP for each dataset. To perform a fair comparison, the architecture of MLP is kept the same for each algorithm. Here, we take only one hidden layer, as one hidden layer is sufficient to classify the datasets shown in Table 1 . The number of neurons in the hidden layer is decided by using the method proposed by [ 25 ]. The number of neurons in the hidden layer is calculated using the formula 2 × N +1, where N is number of relevant features of the dataset. In some cases, the number of hidden neurons taken is 5 × N +1. The architecture of MLP used for each dataset is shown in Table 2 . The values of the controlling parameters of ABC, WOA, MMPSO, MVO, GOA, GA, and GGA-MLP are listed in Table 3 . Various performance metrics such as classification accuracy, specificity, and sensitivity are used to assess the performance of GGA-MLP with respect to the existing state-of-the-art algorithms. The average, best, and standard deviation of classification accuracy, specificity, and sensitivity of the best MLP trained using these metaheuristic algorithms during 30 runs for the given datasets are shown in Tables 4 – 6 , respectively. Data is collected under Windows 10 on Intel core i5-7200U 3.1 GHz processor with 8.00 GB DDR4 and Nvidia GT 940MX 2 GB VRAM. It is evident from Tables 4 and 5 that GGA-MLP gives the highest average and best accuracy as well as specificity for the datasets except Parkinson, QSAR Biodegradation, Drug Consumption: Amyl Nitrite, and Drug Consumption: Ketamine. Despite having low accuracies and specificity on the four datasets, GGA-MLP achieves higher sensitivity as compared to the existing algorithms, as evident from Table 6 , which shows the superiority of GGA-MLP in classifying the positive samples correctly. GGA-MLP also has low standard deviation as compared to existing state-of-the-art algorithms. This shows the robustness of the proposed approach. In Figure 3 , MSE values of MLP trained using ABC, WOA, MMPSO, MVO, GOA, GA, and GGA-MLP for the given datasets are calculated at an interval of 10 iterations and plotted to visualize convergence rate. The convergence curves show that although GGA-MLP takes more time to converge as compared to other metaheuristic algorithms, it avoids getting trapped in local minima. In most of the cases, the performance of GGA-MLP is better than the existing algorithms. To assess the efficacy of MLP trained using GGA-MLP as a classifier, we compare the classification accuracy of GGA-MLP with that of the classifiers built using other machine learning algorithms such as logistic regression, Naïve Bayes, and decision tree, as well as the MLP trained using BP. Similar to decision tree algorithms, BP algorithms like GGA-MLP are also randomized algorithms; every dataset is run 30 times on each of them, and the average, best, and standard deviation of classification accuracy are reported in Table 7 . To prevent overfitting, validation set is used for early stopping during training of logistic regression, Naïve Bayes, and decision tree as well as the MLP using BP. It is clear from Table 7 that GGA-MLP gives the best result in all the cases. However, the standard deviation over 30 runs is least in case of decision tree. From Tables 4 – 7 , it is clear that GGA-MLP performance is better than or comparable to the existing algorithms in classifying input patterns correctly."
} | 3,436 |
29062924 | PMC5640587 | pmc | 4,252 | {
"abstract": "With the developments in metabolic engineering and the emergence of synthetic biology, many breakthroughs in medicinal, biological and chemical products as well as biofuels have been achieved in recent decades. As an important barrier to traditional metabolic engineering, however, the identification of rate-limiting step(s) for the improvement of specific cellular functions is often difficult. Meanwhile, in the case of synthetic biology, more and more BioBricks could be constructed for targeted purposes, but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge. Owing to the lack of steady-state kinetic data for overall flux, balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials. A strategy called targeted engineering is proposed in an effort to solve this problem. Briefly, a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors, substrates and each enzyme will be analyzed systematically. Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays. To demonstrate its practical application, biosynthesis pathways for the production of important products, e.g. chemicals, nutraceuticals and drug precursors, have been engineered in Escherichia coli and Saccharomyces cerevisiae . These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.",
"conclusion": "6 Conclusion and perspectives In these cases, target engineering has shown its potential for metabolic engineering. It is expected to be widely used as there are several advantages compared to traditional metabolic engineering. Firstly, it is much more efficient compared to direct in vivo engineering because we can adjust each component freely and precisely. Instead of constructing hundreds of derivate strains to test their contributions by using genetic methods, e.g. overexpression, deactivation and down-regulation, a series of in vitro assays can be set up easily by adding an exact amount of each component as designed into the system. Secondly, the clear background of the in vitro system makes it useful to explore the maximum potential of the pathway of interest. Each component can be added as designed, which can be very close to the ideal condition to make the pathway work as fast as possible and as smoothly as it can, which is not possible for in vivo engineering. These results help to evaluate the industrial practical possibility of the pathway of interest. Thirdly, the quantified data of each step make it clear for further engineering. The modified targeted proteomics and analysis of intermediates has enriched the targeted engineering because each step can be performed upon the basis of the quantified data. Combinations of these pieces of information will identify the next target. The complexity of a naturally occurring in vivo system often is a major problem for engineering; these systems are products of evolutionary forces and have redundant and often overlapping regulatory elements. During the targeted engineering of a specific pathway, the in vitro system cannot always mimic the in vivo conditions precisely. Interferences occurring in vivo , e.g. the existence of competition or a branched pathway, phosphorylation or acetylation modification of targeted proteins 66 etc., are barriers to pathway engineering. Therefore, an essential interpretation of the targeted pathway is a prerequisite for successful in vitro reconstitution. It should be noted this approach is not suitable for a pathway containing proteins with certain attributes, e.g. poorly soluble, difficult to purify, susceptible to loss of activity in vitro . In addition, the effect of accumulated intermediates on the whole cell system cannot be addressed in the clear background of the in vitro system; the ratio of cofactors of different forms titrated by i n vitro reconstitution is sometimes not consistent as it is in vivo . To construct highly efficient cell factories, both the pathway of interest and the whole cell system should be well balanced. 98 Consequently, by monitoring metabolic status (or metabolomics analysis) and proteomics analysis, this approach could help provide more guidance on metabolic engineering of the whole cell system, such as redox, energy and cofactor metabolism. It is a challenge to express proteins precisely as expected in vivo , which means it is difficult to achieve the exact optimized pathway in vivo . The targeted engineering is still valuable for the in vivo engineering as it can provide quantified data for key factors of the pathway of interest. Targeted engineering will be more useful after a more accurate expression technique is available. Meanwhile, with the widespread application of “-omics” techniques and computational biology techniques, many genome-scale metabolic models have been constructed as tools for various applications, including metabolic engineering, pathway rerouting and systems biology. Combined with targeted engineering, the steady-state kinetic data and the overall flux of a targeted pathway obtained from in vitro reconstitution could facilitate the construction of high-quality metabolic models. The use of a synthetic biology approach in the post-genomic era could artificially design many new pathways for the synthesis of targeted products. The biosynthesis procedures or route of targeted product often can be divided into several synthetic modules upon the basis of their catalyst function. Each synthetic module would be easy to optimize by reconstitution. As a result, modular synthesis of pharmaceutical compounds will likely become the focus of interest, and targeted engineering could be of great benefit to highly efficient modular design and optimization.",
"introduction": "1 Introduction The challenges posed by the energy crisis, environmental degeneration, disease or food shortage and the concerns of achieving sustainable development have prompted great interest in the development of new biological processes and organisms designed for specific purposes. 1 , 2 , 3 Thanks to developments in metabolic engineering and synthetic biology in recent decades, 4 the great potential of microbes as solutions to these dilemmas has entered public knowledge. 5 Metabolic engineering aims to endow cells with improved properties and performance. Synthetic biology could create new biological parts, modules, devices and systems, in addition to re-engineering cellular components and machinery that nature has provided. 6 Through the integration of metabolic engineering and synthetic biology, more efficient microbial cell factories can be constructed to produce biofuels, 7 , 8 biomaterials 9 and drug precursors 10 , 11 from renewable biomass. In the World Economic Forum 2012 (WEF2012), synthetic biology and metabolic engineering were included in the Top 10 Emerging Technologies. With the advent of synthetic biology, especially in the past several years, a few cases involved in the production of pharmaceuticals and new biofuels 12 have been become milestones in this field. The first major practical achievement was the large-scale production of artemisinin by yeast at integrated renewable products company Amyris Inc. 13 Artemisinin, an efficient anti-malarial drug produced by the sweet wormwood plant Artemisia annua , has been used in China for more than 2000 years in the treatment of malaria patients. 14 However, the unstable source of affordable plant-derived artemisinin has resulted in price fluctuations and shortages. 15 As shown in Fig. 1A , Paddon et al. developed a process for the production of artemisinin by fermentation of simple inexpensive carbon substrates using engineered Saccharomyces cerevisiae to produce artemisinic acid, followed by extraction and chemical conversation to artemisinin. The production of artemisinic acid was increased from 1.6 g L –1 to 25 g L –1 . 13 Another landmark work was the total biosynthesis of opioids in yeast. 16 , 17 Opioids, derived from the opium poppy ( Papaver somniferum ), are the primary drugs used for pain management and palliative care. Recently, Smolke's group at Stanford University expressed more than 20 enzymes derived from rodents, plants and bacteria in a engineered host and, finally, realized the complete biosynthesis of opioids from glucose. 18 In these cases, synthetic biological approaches have been used to optimize both the host and pathways to maximize the production of targeted products. Although synthetic biology allows us to freely manipulate the components (e.g. promoters, enzymes, modules, etc.), just like building blocks, an optimal pattern has to be selected from millions of combinations ( Fig. 1B ). A common approach, for example, is to investigate as many mutants as possible; however, if a high-throughput method cannot be generated or a large mutant selection is too expensive, it would be difficult to obtain satisfactory results. It is worth noting that the formation of artemisinic acid requires enormous manpower and financial resources. 19 , 20 Fig. 1 Prospects and challenges of synthetic biology in the construction of high-efficiency microbial cell factories. (A) High-efficiency biosynthesis of the artemisinin precursor in yeast. The genes expressed encode the following enzymes: AtoB, acetoacetyl-CoA thiolase; ERG13, HMG-CoA synthase; tHMG1, truncated HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; MVD1, mevalonate diphosphate decarboxylase; Idi, isopentenyl diphosphate (IPP) isomerase; IspA, farnesy diphosphate (FPP) synthase; ADS, amorpha-4,11-diene synthase; CYP71AV1, cytochrome P450 enzyme that converts amorphadiene to artemisinic alcohol; CPR, cytochrome P450 reductase; CYB5, cytochrome b5; ADH1, artemisinic alcohol dehydrogenase; ALDH1, artemisinic aldehyde dehydrogenase. CYP71AVA1, CPR1, CYB5, ADH1 and ALDH1 derived from A. annua could oxidize amorphadiene to artemisinic acid. Genes colored light blue are derived from E. coli , dark blue genes are derived from S. cerevisiae , red genes is derived from Staphylococcus aureus , purple genes are derived from A. annua . (B) Challenges to the optimization of a biosynthetic module. The synthetic biology components, such as kinds of promoters and enzymes, could be constructed like building blocks, and the optimal pattern have to be selected from millions of combinations. Traditional metabolic engineering has made great advances in the optimization and innovation of industrial fermentation, including the biosynthesis of a taxol precursor in microbes, 21 conversion of lignocellulosic biomass to ethanol 22 and application of amino acid-producing bacteria. 23 The heterologous synthesis of a taxol precursor in Escherichia coli was one of the most famous works in the field of metabolic engineering. Taxol is a potent anticancer drug produced by the Pacific yew tree Taxus brevifolia . 24 Ajikumar et al. reported integration of a native upstream methylerythritol phosphate (MEP) pathway and a heterologous downstream terpenoid-forming pathway allowed taxadiene, the first committed taxol intermediate, to be obtained in large amounts from E. coli by fermentation. 21 In the bioenergy field, there are improved production rates of advanced biofuels, including butanol, hydrocarbons and terpene-based biofuels. 25 , 26 , 27 However, one important challenge for traditional metabolic engineering is the identification of gene targets of major importance for the improvement of specific cellular functions. 28 Additionally, owing to the lack of biochemical information and genetic background of the targeted metabolic pathways, many engineering works have not achieved the expected results. A strategy called targeted engineering was proposed in an attempt to overcome these problems and challenges. For this strategy, the biosynthetic pathway is reconstituted in vitro and then the contributions of cofactors, substrates and enzymes are analyzed systematically. Subsequently, in vivo engineering could be guided by the information gained from in vitro assays. This approach might offer some opportunities to create cell factories based upon constructed biological components. Here, we present a review of targeted engineering and its application."
} | 3,130 |
31260266 | null | s2 | 4,254 | {
"abstract": "Open microfluidic capillary systems are a rapidly evolving branch of microfluidics where fluids are manipulated by capillary forces in channels lacking physical walls on all sides. Typical channel geometries include grooves, rails, or beams and complex systems with multiple air-liquid interfaces. Removing channel walls allows access for retrieval (fluid sampling) and addition (pipetting reagents or adding objects like tissue scaffolds) at any point in the channel; the entire channel becomes a \"device-to-world\" interface, whereas such interfaces are limited to device inlets and outlets in traditional closed-channel microfluidics. Open microfluidic capillary systems are simple to fabricate and reliable to operate. Prototyping methods (e.g., 3D printing) and manufacturing methods (e.g., injection molding) can be used seamlessly, accelerating development. This Perspective highlights fundamentals of open microfluidic capillary systems including unique advantages, design considerations, fabrication methods, and analytical considerations for flow; device features that can be combined to create a \"toolbox\" for fluid manipulation; and applications in biology, diagnostics, chemistry, sensing, and biphasic applications."
} | 307 |
39650726 | null | s2 | 4,255 | {
"abstract": "Natural biomolecular systems have evolved to form a rich variety of supramolecular materials and machinery fundamental to cellular function. The assembly of these structures commonly involves interactions between specific molecular building blocks, a strategy that can also be replicated in an artificial setting to prepare functional materials. The self-assembly of synthetic biomimetic peptides thus allows the exploration of chemical and sequence space beyond that used routinely by biology. In this Review, we discuss recent conceptual and experimental advances in self-assembling artificial peptidic materials. In particular, we explore how naturally occurring structures and phenomena have inspired the development of functional biomimetic materials that we can harness for potential interactions with biological systems. As our fundamental understanding of peptide self-assembly evolves, increasingly sophisticated materials and applications emerge and lead to the development of a new set of building blocks and assembly principles relevant to materials science, molecular biology, nanotechnology and precision medicine."
} | 282 |
39650726 | null | s2 | 4,256 | {
"abstract": "Natural biomolecular systems have evolved to form a rich variety of supramolecular materials and machinery fundamental to cellular function. The assembly of these structures commonly involves interactions between specific molecular building blocks, a strategy that can also be replicated in an artificial setting to prepare functional materials. The self-assembly of synthetic biomimetic peptides thus allows the exploration of chemical and sequence space beyond that used routinely by biology. In this Review, we discuss recent conceptual and experimental advances in self-assembling artificial peptidic materials. In particular, we explore how naturally occurring structures and phenomena have inspired the development of functional biomimetic materials that we can harness for potential interactions with biological systems. As our fundamental understanding of peptide self-assembly evolves, increasingly sophisticated materials and applications emerge and lead to the development of a new set of building blocks and assembly principles relevant to materials science, molecular biology, nanotechnology and precision medicine."
} | 282 |
37582974 | PMC10427580 | pmc | 4,258 | {
"abstract": "Highlights \n The addressable electrical contact structure enables the multifunctional epidermal interface with an all-in-one function of sense, recognition, and transmission, which realizes high flexibility and high-precision touch detection. The multifunctional epidermal interface achieves superior waterproofness and is constructed enough thin to be bent freely, which is not as rigid, bulky, and thick as common interactive electronic device. The bending-insensitive characteristic facilitates accurate and stable human–machine interactions, which provides a key foundation for intelligent prostheses and super-soft robots. \n Supplementary Information The online version contains supplementary material available at 10.1007/s40820-023-01176-5.",
"conclusion": "Conclusions In the work, we have taken initial inspiration from the biological sensory system and made a breakthrough to construct a highly bending-insensitive, unpixelated and waterproof epidermal interface (BUW epidermal interface) for the artificial touch sensing system. CNT and MC were combined to achieve complementary advantages for preparing sensitive material with high electrical property and high stability. The addressable electrical contact structure with the satisfactory sensitive material was designed to enable the BUW epidermal interface with an all-in-one function of sense, recognition, and transmission. Without large-scale integration of sensing pixels, the BUW epidermal interface overcame the performance contradiction and realized high flexibility and high-precision touch detection. Due to the efficient addressable electrical contact structure, the BUW epidermal interface could achieve superior waterproofness and was prepared enough thin to be bent freely, which was not as rigid, bulky, and thick as common waterproof IE devices. Regardless of whether being flat or bent, the BUW epidermal interface could be conformably attached to the human skin as a touch operation platform with rapid response and recovery time of both < 8 ms and high durability of > 20,000 cycle tests. It eliminated the need for baseline offset or relationship redefinition between signals and instruction, which provided great advantages as a highly stable and flexible IE device to be mounted on the curved body surface for free, comfortable, and unrestrained HMIs. The BUW epidermal interface possessed the accurate spatiotemporal dynamic recognition capability like the one of the biological touch sensory system. The highly regional differentiated recognition capability and scalability, and versatility ensured that the BUW epidermal interface could be designed into as-needed shapes for diverse interactive control, including controlling chess piece positioning, the realistic tank movement, and the manipulation of virtual gun barrel. In particular, the BUW epidermal interface achieved the “cut-and-paste” character that allowed it to be corresponding size and mounted on the palm for diverse HMIs. The artificial touch sensing system with the BUW epidermal interface will enable things with an improved reproduction of tactile sense, which undoubtedly provides a major scientific and technological breakthrough for the accurate reproduction of human sense toward metaverse. In the context of real-world interactive situations, the skin surface areas can not only be bent but also stretched. Therefore, for achieving the stretchability of the BUW epidermal interface, stretchable polymer substrates, such as poly(dimethylsiloxane), polyurethane, and Ecoflex silicone elastomer, may be viable alternatives. Furthermore, the combination of functional materials with stretchable substrate requires further exploration and research to realize stable signal output and recognition of the stretchable epidermal interface during the stretching process. The BUW epidermal interface based on the addressable electrical contact structure can effectively sense and recognize one touch point per time. In certain interactive applications, multi-touch sensing is also needed. It requires innovation in structural design and material exploration. Also, the simplicity and effectiveness of the device’s structure still should be deserved more attention. In addition, the exploration of body-integrated devices for real-time monitoring will dramatically benefit patients and remarkably improve their quality of life. Hence, one of the key challenges lies in the need for implantable and degradable materials. In the future, the combination of various functional materials and novel structures will enable the epidermal interface to be stretchable, multi-point touchable, implantable, and degradable.",
"introduction": "Introduction Interactive electronic devices (IE devices) in the metaverse enable users to enhance the quality of interactions and create real-time closed-loop feedback through an intuitive interface [ 1 – 7 ]. As if creating a virtual world, humans still can use their fingers and eyes to interact with the virtual world through IE devices, which makes the life of the avatar more real and the interaction more direct [ 8 – 11 ]. Metaverse reconstructs a new scene of the human–machine relationship, which gives an immersive interactive experience at any time as needed, such as exploring in a humid jungle or surfing on the wet sea. To resist humid surroundings, such as rain and sweat, IE devices are tightly encapsulated to obtain waterproofness [ 12 – 15 ]. This way generally makes IE devices to be rigid, bulky, and thick. Owing to the inherently soft human tissue surfaces, a large mismatch will be generated between IE devices and the human tissue surface [ 16 – 18 ]. To provide a smooth and relatively comfortable touch interaction, the IE devices need to be flexible and fit well on the skin surface [ 19 , 20 ]. In reality, the current IE devices may still encounter malfunction due to bending deformation, making the sensing units out-of-operation or electrodes connection disconnected [ 21 – 23 ]. Thus, IE devices should have high flexibility so that they can be conformal with soft skin and continuously function stably even if they are deformed due to wearing [ 24 – 26 ]. Furthermore, the acquisition of high performance enables IE devices to accurately convert human intentions into computer-recognizable signals, which is critical to the breakthrough development of the metaverse. Accurate and real-time information measurement using IE devices, such as stretchable transistors, haptic interfaces, and artificial skin [ 27 – 31 ], attached to the human body was crucial for human–machine interactions (HMIs). Through mutually non-overlapping capacitive signals, a flexible magnetic field sensor could identify the magnitude and direction of the magnetic field for contactless measurement and interaction [ 32 ]. To render entire IE devices flexible, efficient alternatives were to adopt soft functional materials to make sensing pixels flexible directly, which could be attached to the human skin as a touch operation platform [ 33 – 37 ]. To accurately recognize touch location, large-scale integrations of sensing pixels were common methods [ 38 – 41 ], which resulted in the overall stiffness of IE devices. To turn hard IE devices into flexible ones, alternatives were to design shape-variable conductive wires to connect sensing pixels by reducing pixel density [ 42 – 44 ]. Thus, high flexibility and high-precision touch detection seemed to be irreconcilable [ 45 ]. In addition, as sensing signals varied when flexible IE devices were mounted on the human body [ 23 , 36 ], baseline offset or relationship redefinition between signals and instructions was generally needed while it was frustrating in HMIs. Here, we introduce a highly bending-insensitive, unpixelated, and waterproof epidermal interface (BUW epidermal interface) to create conformal human–machine integration for free, wearable, and accurate interactions. The BUW epidermal interface mimics the structure and function of the mechanoreceptors in the biological touch sensory system, which can convert the touch information into the mechanosensitive signal for identification and differentiation. An addressable electrical contact structure is proposed to enable the BUW epidermal interface to precisely recognize touch location without large-scale integration of sensing pixels. The BUW epidermal interface is composited of ultrathin polyethylene terephthalate (PET) film, carbon nanotube (CNT), and methylcellulose (MC). By adopting a hierarchical assembly, the ultrathin PET film is served as an outside substrate, which endows the BUW epidermal interface with waterproofness and maintains the overall softness of the device. The excellent conductivity of the CNT makes the BUW epidermal interface possesses a rapid response time of < 8 ms and high-precision touch detection, which ensures accurate identification of users' control intent into stable and effective interactions. The MC provides a certain viscosity for the CNT to adhere well to the PET film, which guarantees that the signal transmission does not change even if the BUW epidermal interface is bent or deformed. As a proof-of-concept, this bending-insensitive characteristic is demonstrated by conformal integration of the BUW epidermal interface onto the people’s palms for virtually beating chime freely. Meanwhile, the unpixelated structural design strategy presented here facilitates the high-density touch sensing capability and the wide space of sensing area, of which the functional instructions can be programmable at will. Due to the practical scalability and versatility of the design principle, the BUW epidermal interface can be designed for special structures with different shapes for diverse interactive control toward the metaverse. The BUW epidermal interface not only proposes an unpixelated and waterproof structure for imitating the complicated biological touch sensory system but also innovatively exhibits the highly bending-insensitive characteristic to realize an artificial touch sensing system for broad relevance to conformally integrated electronics.",
"discussion": "Results and Discussion Operation Principle and Design of the BUW Epidermal Interface The concept behind the biological touch sensory system and the artificial touch sensing system using the BUW epidermal interface is shown in Fig. 1 . The biological touch sensory system detects external mechanical stimulation by converting them into receptor potentials using different types of cutaneous receptors (Fig. 1 a), including Meissner corpuscle, Ruffini corpuscl, Merkel disk, and Pacinian corpuscle [ 46 ]. The receptor potentials are processed by synaptic transmission and spike generation. Different signals are gathered by nerve fibers. The resulting feedback conducts according to the brain, which judges the signals and makes decisions. Thus, the human body produces tactile perception for manipulating things by the corresponding feedback signals. To construct a system with an artificial touch sensing function, an artificial touch sensing system was designed that mimicked the human tactile recognition using the BUW epidermal interface, signal transmission, and processing system (Fig. 1 b). The BUW epidermal interface sensed and recognized the external mechanical stimulation and transmitted the mechanosensitive signal to a microcontroller unit (MCU). Then, an analog-to-digital conversion (ADC) module in the MCU converted the analog signal into a digital signal. Finally, the digital signal was transmitted to a signal processor for making efficient decisions of HMIs. In the artificial touch sensing system, the BUW epidermal interface played a vital role in replacing various mechanoreceptors and generating mechanosensitive signals, which could sense and recognize the mechanical stimulation and transmit the mechanosensitive signal. The BUW epidermal interface was based on the addressable electrical contact structure. It consisted of two ultrathin PET films with the CNT/MC sensitive material, which were assembled in a mirror-symmetrical way. In the design, the BUW epidermal interface was thin enough to realize conformal integration with the human body. Figure 1 c shows that the BUW epidermal interface was fabricated using the CNT/MC-based sensitive material layer, PET film-based protective layer, and spacer-based insulator layer. The microscopic images of the BUW epidermal interface clearly presented that the thickness of the addressable sensing layers was 27 μm (Fig. S2), where the thickness of the PET film-based protective layer was 20 μm (Fig. S3). Thus, the thickness of the CNT/MC-based sensitive material layer, which was part of the addressable sensing layer subtracted the PET film, was 7 μm. During encapsulation, the two addressable sensing layers were supported by a spacer-based insulative layer with the thickness of 160 μm. As the total thickness of the BUW epidermal interface was only 214 μm, which endowed the BUW epidermal interface with an excellent flexibility (Fig. 1 d). The field emission scanning electron microscopy (FESEM) image presented the uniformly dispersed CNT/MC-based sensitive material layer, where the CNT was pointed out by a yellow arrow (Fig. 1 e). It should be noted that CNT had good electrical conductivity but was not sticky. Since MC had good dispersion and viscosity, it could act as a binder and was mixed with CNT dispersion to make the CNT/MC-based sensitive material layer both conductive and sticky. Thus, the CNT/MC-based sensitive material layer could adhere well to the PET film no matter in the flat state or bending state. As a comparison, another epidermal interface with the thickness of 2 mm was fabricated. When being bent, the thick epidermal interface generated undesirable creases, which resulted in an inherent mismatch in functions (Fig. S4). This situation was similar to one of the common devices based on thicker and less elastic materials, which could not be used for conformal human–machine integration of free, wearable, and accurate interactions. Fig. 1 Biological touch sensory system and artificial touch sensing system. a Biological touch sensory system, including the skin, relevant mechanoreceptors, and neural system. b Artificial touch sensing system based on the BUW epidermal interface, ADC module, and signal processor. c Addressable electrical contact structure of the BUW epidermal interface composed of CNT/MC-based sensitive material layer, PET film-based protective layer, and spacer-based insulative layer. d Photograph of the BUW epidermal interface in the bending state, showing excellent bendability. e FESEM image of CNT/MC-based sensitive material layer, where the CNT was pointed out by a yellow arrow. (Color figure online) The designed addressable electrical contact structure and its equivalent circuit for the working principle of the BUW epidermal interface were explained in the electronic aspect (Fig. S5). The addressable sensing layer could virtually represent a series of resistive units, which was equivalent to the integration of multiple sensing pixels (Fig. S5a). The spacing area between the touch areas was similar to the air switch between the upper and bottom resistive units. When an external mechanical stimulation was applied at a touch location of the BUW epidermal interface, the corresponding air switch (e.g., S 2 in Fig. S5a) closed. The BUW epidermal interface would generate a response resistance ( R total ), which was expressed as: 1 \\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}$$R_{{{\\text{total}}}} = R_{{{\\text{u1}}}} + R_{{{\\text{u2}}}} + R_{{{\\text{b1}}}} + R_{{{\\text{b2}}}} + R_{{{\\text{c2}}}}$$\\end{document} R total = R u1 + R u2 + R b1 + R b2 + R c2 where the R u and R b were the resistance of the virtual resistor units on the upper and bottom addressable sensing layers, respectively, and R c was the contact resistance between the upper and bottom addressable sensing layers. Thus, the BUW epidermal interface could sense whether the external mechanical stimulation was applied. As the BUW epidermal interface was designed to be long-strip shape, any touch location of the BUW epidermal interface could respond to the external mechanical stimulation and generate a corresponding response resistance (Fig. S5b). When the external mechanical stimulations were applied at two touch locations of the BUW epidermal interface with the interval time Δ t , the corresponding air switches (e.g., S 2 and S 4 in Fig. S5b) would close and open sequentially. The corresponding but different response resistances of the BUW epidermal interface were generated to make a dynamic mechanosensitive signal. According to the response resistance of the mechanosensitive signal, the BUW epidermal interface could recognize the touch location, where the external mechanical stimulation was applied. The involving resistive units of the upper and bottom addressable sensing layers could transmit the electrical signal to the back-end circuit for processing and analysis. Therefore, the designed addressable electrical contact structure endowed the BUW epidermal interface with unpixelated sensing and recognition, which did not require large-scale integrations of sensing pixels. It greatly simplified the structure of the device so that the operation was efficient and convenient, thus reducing the difficulty of the fabrication process. More prominently, as the BUW epidermal interface was ultrathin, the external mechanical stimulation could still be sensed and recognized even when the BUW epidermal interface was bent (Fig. S5c, d). Due to the presence of spacers, the upper and bottom addressable sensing layers were still separated even if the BUW epidermal interface was bent. Once the external mechanical stimulation was applied, the upper and bottom addressable sensing layers would contact, causing the BUW epidermal interface to generate a response resistance ( R′ total ). The surface deformation ( ε ) caused by bending the BUW epidermal interface was calculated using this formula: 2 \\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}$$\\varepsilon = h/2{r}$$\\end{document} ε = h / 2 r where h was twice the distance of the surface from the neutral plane and r was the curvature radius. Because the BUW epidermal interface was ultrathin, bending the BUW epidermal interface only made very small deformation of the upper and bottom addressable sensing layers (Fig. S5). This very small deformation would not further cause an observable change in the response resistance of the BUW epidermal interface. In this way, when the external mechanical stimulation was applied at the same touch location, the response resistance of the BUW epidermal interface would be almost the same, regardless of whether being flat or bent. Therefore, the response resistance of the BUW epidermal interface was insensitive to bending and could be used to accurately sense and recognize external mechanical stimulation. This bending-insensitive characteristic eliminated the need for baseline offset or relationship redefinition between signals and instructions, even if the BUW epidermal interface was deformed by wearing, which would facilitate comfortable and free HMIs. It is worth noting that the biological mechanoreceptor possesses a threshold value for the input mechanical stimulation in a biological touch sensory system. The body cannot produce sensation when the input mechanical stimulation does not exceed the threshold value. In the artificial touch sensing system, the BUW epidermal interface also had a threshold value of external mechanical stimulation (Fig. S6). After the pressure of the external mechanical stimulation was larger than 4.5 kPa, the BUW epidermal interface could accurately sense and stably recognize the external mechanical stimulation. In the following tests, the pressure of the external mechanical stimulation was about or large than 6 kPa, unless otherwise noted. For common IE devices on standby, they were running at full power and the energy was always consumed regardless of whether external mechanical stimulation was applied, which would bring potential uncertainty or shortened life to long-term stable operation [ 12 , 23 ]. However, when there was no external mechanical stimulation, the upper and bottom addressable sensing layers of the BUW epidermal interface were separated. Thus, the BUW epidermal interface did not consume energy on standby, which could improve the comprehensive energy utilization efficiency. Performance and Characteristic of the BUW Epidermal Interface Figure 2 a shows the change in the response resistance of the BUW epidermal interface when external mechanical stimulation was applied at different touch locations. It could be found that the response resistance of the BUW epidermal interface increased almost linearly with the change of the touch location. It was mainly because the response resistance of the BUW epidermal interface was affected by the involving resistance of the addressable sensing layers (Fig. S7), which was linear with the length of the addressable sensing layer. In addition, regardless of the different lengths of the BUW epidermal interface, such as 50, 100, or 200 mm, the linear relationships between the response resistance and the touch location were almost the same. The longer the effective working length of the BUW epidermal interface was, the more abundant the touch location became. To test the response resistance of the bending BUW epidermal interface, the BUW epidermal interfaces were winded diagonally around cylinders with different radiuses (Fig. 2 b, c). It could be found that the response resistance was relatively linear with the touch location no matter how the radiuses of the cylinders are, which was important to accurately recognize the touch location. When touching the same location, the response resistance of the bending BUW epidermal interface was almost the same as that of the flat BUW epidermal interface. This was because the BUW epidermal interface was so thin that the bending strain was too small to change the response resistance. In a dynamical loading-unloading test, the mechanosensitive signals were also almost in the same change way no matter whether the BUW epidermal interface was in the flat or bending state (Fig. S8). This bending-insensitive characteristic ensured that the bending BUW epidermal interface could stably recognize the touch location as the case in the flat state. It eliminated the need for baseline offset or relationship redefinition between signals and instruction, which provided great advantages as a highly stable and flexible IE device to be mounted on the curved body surface for comfortable and unrestrained HMIs. Fig. 2 Bending-insensitive, spatiotemporal dynamic, and waterproof characteristics of the BUW epidermal interface. a Relationship between response resistance and touch location of the BUW epidermal interface with different lengths. b , c Change in response resistance versus touch location when the BUW epidermal interface was winded diagonally around cylinders with different radiuses. d Diagrams of two biological synapses and the BUW epidermal interface subjected to an external mechanical stimulation, which was applied at different location. e , f Repetitive responses of the BUW epidermal interface in the flat or bending state when applying the spatiotemporally dynamic stimulations. g Relationship between response resistance and touch location of the BUW epidermal interface in dry surroundings and humid surroundings. h Photographs and multiple cyclical tests of the BUW epidermal interface in (i) dry and (ii) humid surroundings In the biological touch sensory system, even when the skin tissue was bent or deformed, the basic dynamic characteristic signals of external mechanical stimulation could still be transmitted through the biological synapses and the nerve fibers. Thus, a recognizable and differentiated dynamic logic in the neural network would be established to obtain spatiotemporal signals of different touch locations (Fig. 2 d). In the artificial touch sensing system, the BUW epidermal interface could also possess the spatiotemporal dynamic recognition capability even if in the bending state. In the flat state, the response resistance of the BUW epidermal interface would rapidly rise up to a certain value and remained over time when an external mechanical stimulation was applied at a touch location, which was at 20% of the length (Fig. 2 e). Until the external mechanical stimulation was released, the response resistance quickly disappeared. When the external mechanical stimulation was applied at another touch location, which was at 80% of the length of the BUW epidermal interface, the response resistance arose with corresponding magnitude for the recognition. Once the touch location was changed to the original one, the magnitude of the response resistance would be changed accordingly. In this way, the external mechanical stimulation at different times and touch locations could be accurately sensed and recognized by the BUW epidermal interface. The spatiotemporal dynamic stimulation could be also recognized by the bending BUW epidermal interface (Fig. 2 f). By continuous and repeated mechanical stimulation, the response resistance could vary according to the different touch locations over time. When the external mechanical stimulation with the interval time of 1.02 s was applied at the relative locations at 20% and 80% of the length of the bending BUW epidermal interface, the corresponding response resistance in periods was generated and kept within a certain magnitude range. It could be found that the response resistance of the BUW epidermal interface was highly distinguishable from the spatiotemporal dynamic mechanical stimulation. The results manifested that the response signal could reflect well about the spatial and temporal feature of the external mechanical stimulation no matter whether the BUW epidermal interface was in the flat or bending state. The geometrically hierarchical response signal of the BUW epidermal interface realized the sensing and recognition function of an IE device consisting of numerous sensing elements, but its actual structure did not require so many sensing elements. For the utilization of the BUW epidermal interface in the human–machine interaction system, the response time, response stability, and response frequency were also crucial factors to be considered. Since the BUW epidermal interface was thin enough, mechanical stimulation could cause the upper and lower layers to contact rapidly. Once the mechanical stimulation was removed, the upper and lower layers could be quickly separated, so the response time would be extremely fast. By applying external mechanical stimulation on the BUW epidermal interface, the rapid response time and recovery time of both < 8 ms could be tested, providing a significant advantage for the real-time feedback control system (Fig. S9). As the device’s structure was unified and the CNT/MC-based sensitive material layer was homogeneous, the sensing characteristics of each location of the BUW epidermal interface were the same. Therefore, the response time did not vary according to the touch locations. Due to the design of the addressable electrical contact structure, the BUW epidermal interface could realize unpixelated sensing and recognition (Fig. S10). It could be found that the change of the response curve was continuous. The unpixelated sensing characteristic meant that there was no independent pixel on the BUW epidermal interface so that any locations could be recognized well and correspondingly produce specific response. Comparing with the pixelated device, the BUW epidermal interface could be fully flexible and not affected by the wiring topology. In addition, only two electrodes were required to accurately sense and recognize the location, while most pixelated devices required many pixel routings with numerous electrodes. Furthermore, the cyclic mechanical stimulation (6 kPa and 1 Hz) was applied on the BUW epidermal interface for long-term operation analysis (Fig. S11). During a long cycle test (> 20,000 times), the response resistance was slightly changed (~ 9%). It was considered that the compression position and depth of the customized actuator slipped slightly as time. It could be found that the BUW epidermal interface was still capable of producing a response curve with regular undulations. Therefore, the BUW epidermal interface possessed excellent long-term stability and durability for potential wearable applications. Furthermore, different touch response frequencies were tested when the BUW epidermal interface was in the bending state (Fig. S12). The result showed that within a routine frequency range, although the frequency of the external mechanical stimulation changed, the response resistance of the BUW epidermal interface was relatively stable and there was no distortion or frequency loss. Therefore, the BUW epidermal interface could be used for stable and continuous touch interactions without re-correction in the signaling processes. All these tests manifested that the BUW epidermal interface possessed the potential for dynamic consecutive sensing and recognition of external mechanical stimulation, which could be attached on the various parts of the human body. To consider potential applications in wet operating environments (e.g., humid jungle environment or surfing at sea), the waterproofness was a crucial characteristic. Since the upper and bottom addressable sensing layers were sealed together face to face and the electrodes were completely encapsulated with the double-sided tape, the designed BUW epidermal interface was endowed with excellent waterproofness and possessed superior reproducibility and good stability whenever being used in dry and humid surroundings. Figure 2 g presented the relationship between the response resistance and the touch location in dry and humid surroundings. It could be found that the response resistance of the BUW epidermal interface remained almost constant in surroundings with different humidity conditions. Due to the strong adhesive force of the double-sided tape, the addressable electrical contact structure of the BUW epidermal interface effectively isolated the CNT/MC-based sensitive material layer from water or sweat. In this way, the BUW epidermal interface possessed waterproofness and flexibility, not as rigid, bulky, and thick as common waterproof IE devices. During a dynamic test, the external mechanical stimulation was applied on the BUW epidermal interface in dry and humid surroundings (Fig. 2 h). Multiple cyclic tests were performed in the same touch location, which was well reflected by almost the same response resistance. In addition, even when being submerged in water, the BUW epidermal interface could still recognize the external mechanical stimulation and possessed good reproducibility (Fig. S13). This response resistances were almost unaffected whether the BUW epidermal interface was in dry or underwater surroundings. Thus, the back-end information processing did not need to be recalibrated. The results confirmed that the BUW epidermal interface had good waterproofness, which could continue to function even in underwater surroundings. Common IE devices were designed with complex connection lines between pixelated sensing elements. They would be completely disabled when partially damaged, resulting in the collapse of the entire interactive system. Attributing to the addressable electrical contact structure, even if part of the BUW epidermal interface was cut, the two cuttable rest would still be able to sense, recognize, and transmit touch information (Fig. S14a). Since the BUW epidermal interface mainly consisted of CNT/MC-based sensitive material, PET film, and double-sided tape, all of which were cuttable, the as-fabricated device could be conveniently cut off with the scissor. After cutting, one section of the BUW epidermal interface consisting of the remaining conductive sensing layer and the electrodes still retained the functions. The other section consisting of the terminal portion and the re-added electrodes could restore the functions. The test result showed that after part of the BUW epidermal interface was cut off, the linear relationship between the response resistance and the touch location was almost identical to that of the original one (Fig. S14b). Therefore, the BUW epidermal interface could be easily cut into different lengths depending on the need for personalized HMIs. Due to the unpixellated working characteristic, the BUW epidermal interface could be virtually divided into multiple working segments for the needs of intelligent social HMIs. A dynamic cyclic mechanical stimulation was applied at the different virtual working segments of the BUW epidermal interface for the test (Fig. S15). The result indicated that each virtual working segment could well sense and recognize the mechanical stimulation. Multifunctional Interactive Applications of the BUW Epidermal Interface With the rapid development of virtual reality (VR) technology, various types of IE devices were developed to enrich the entertainment experience of relaxation and decompression. The interactive signals would be fluctuating due to bending common IE devices to fit the curved surface of the soft human body so that inaccurate commands were generated to make a constrained experience. Herein, as a proof-of-concept, the BUW epidermal interface realized conformal human–machine integration to achieve the comfortable and unconstrained interaction of virtual chimes. Due to the highly flexibility, unique bending-insensitive characteristic, and excellent waterproofness, the BUW epidermal interface could be fixed on the curved surface of a soft palm to make the palm as an interactive platform for HMIs (Fig. 3 a). An interactive VR system, which consisted of a BUW epidermal interface, an analog-to-digital conversion (ADC) module, a microcontroller unit (MCU), a universal serial bus (USB) interface, and computer software, was designed and developed (Fig. 3 b). A linear BUW epidermal interface with an effective working length of 140 mm was designed and mounted on the palm to function. When the other finger touched the BUW epidermal interface, the touch location was deformed to make the specific upper and bottom addressable sensing layers contact to quickly generate a mechanosensitive signal. Next, the MCU received and processed the recognizable mechanosensitive signal. Finally, the signal was judged and transmitted to the Unity development application that controlled the virtual chimes through the universal serial bus. It was considered that the size of the pressing area would be different in different touches and the touching width of a finger was about 10 mm. In the demonstration, the linear BUW epidermal interface was virtually divided into fourteen working segments with a width of 10 mm, namely from L 1 to L 14 (Fig. 3 c). Thus, touching a specific working segment of the BUW epidermal interface from left to right could accurately beat the corresponding chime, namely from B 1 to B 14 . Figure 3 d shows that the response resistances of the working segments were highly different from each other, which was beneficial to efficiently and consistently performing the HMI of beating chimes. When a working segment of the BUW epidermal interface was touched, a virtual hammer would move to the corresponding location to beat the chime in the VR interactive system (Fig. 3 e). It was like that fourteen couples of virtual mechanoreceptors and synapses were embedded into the BUW epidermal interface, each of which could sense and convert external mechanical stimulation into the mechanosensitive signal. After abundant trials and tests, the addressable sensing layers were divided into specific intervals, allowing the BUW epidermal interface to accurately sense touch and recognize its location. For example, when a fingertip touched a working segment, the BUW epidermal interface could sense the touch and recognize the touch location so that the corresponding chime was triggered and belled. As a conceptual wearable verification, some typical examples of interactively beating chimes were conducted by touching the BUW epidermal interface, which was fixed on the soft palm (Fig. 3 f). The BUW epidermal interface could bend and deform with the soft palm. As the bending-insensitive and wearable characteristic, the BUW epidermal interface could work normally without baseline compensation or signal verification. Even if the BUW epidermal interface was bent, it could still accurately sense and recognize each touch and stably trigger the belling of the corresponding chime. In addition, the BUW epidermal interface was capable of realizing no energy consumption when there was no external mechanical stimulation, which paved the way for long-term and effective use for entertainment activities in the VR interactive system. A vivid demo using the BUW epidermal interface to interact with the virtual chimes was presented in Movie S1. Fig. 3 BUW epidermal interface for beating chime in the interactive VR system. a Schematic of the BUW epidermal interface installed on the palm. b Flowchart of HMI based on the BUW epidermal interface for the Unity application development. c Touching different working segments of the BUW epidermal interface corresponding to beating different chimes. d Typical response resistances when touching different working segments. e Touching a working segment of the BUW epidermal interface to trigger the corresponding chime. f Typical photos of beating the virtual chime by touching the BUW epidermal interface, where the yellow rectangles indicated the BUW epidermal interface fixed on the soft palm and the yellow ellipses indicated the beaten chime. (Color figure online) The designed addressable electrical contact structure greatly simplified the device structure and reduced the preparation process, thereby improving the scalability and versatility of the BUW epidermal interface. Thereupon, the BUW epidermal interface was designed into square, plus-shaped, and hexagon-shaped structures, which could be well applied to the corresponding interactive applications (Fig. S16). The representative demonstration of controlling chess piece positioning was introduced. Figure 4 a shows the movement trajectories of the white and black chess pieces on a virtual chessboard in a competition between humans and machines. In the course of the chess piece movement, the white chess piece moved from the bottom left corner to the upper right corner and the black chess piece moved from the upper right corner to the bottom left corner. The white and black chess pieces in the VR space were created, which represented the controllable and programmable multivariant movement for chess piece positioning. Figure 4 b presents the square BUW epidermal interface, which could be virtually divided into multiple working segments, and their corresponding biomimetic synapses. The highly regional differentiated recognition capability was realized by the square BUW epidermal interface (Fig. S17). The working segments at the bottom and right part of the square BUW epidermal interface were used to control the movement of white chess piece (the cyan arrow in Fig. 4 b) and the other working segments were used to control the movement of black chess piece (the red arrow in Fig. 4 b). The horizontal part controlled the horizontal movement of chess pieces and the other part controlled the movement of chess pieces along the vertical axis. When the finger touched the X B , X C , X D , Y 2 , Y 3 , Y 4 , and Y 5 working segments of the square BUW epidermal interface, the response resistances of the corresponding working segments of the square BUW epidermal interface were rapidly generated (Fig. 4 c). As a result, the white chess piece moved to the corresponding position on the virtual chessboard. Similarly, touching the X J , X K , X L , Y 10 , Y 11 , Y 12 , and Y 13 working segments of the square BUW epidermal interface would make the black chess piece move to the corresponding position on the virtual chessboard. As a verification, this demonstration of the virtual white and black chess pieces’ movement by the square BUW epidermal interface was designed (Fig. 4 d). In the developed interactive VR game-playing entertainment, touching different working segments would accurately make the virtual white and black chess pieces move to the corresponding position. A vivid demo using the square BUW epidermal interface to interact with the virtual chess pieces’ movement was presented in Movie S2. Owing to the facile square BUW epidermal interface, the human–machine interaction system based on touch not only provided a simplified and universal solution for capturing the human touch position but also introduced a strategy of avoiding the potential issues of the integration of numerous sensing units for the elimination of the complex interconnections and the signal crosstalk. Fig. 4 Square BUW epidermal interface for controlling chess piece position movement. a Diagram of the chess board and the movement trails of the white and black chess pieces. b Structure feature of the working segments of the square BUW epidermal interface and their corresponding biomimetic synapses. c Typical response resistances of the square BUW epidermal interface when touching different working segments. d Typical photos of different movement processes of virtual chess pieces by touching the square BUW epidermal interface, where red arrows indicated the chess piece positions and the blue arrows indicated the working segments of the square BUW epidermal interface. (Color figure online) Various special shapes of the BUW epidermal interface were adapted to interactive entertaining applications to achieve an enhanced users’ experience. As an example, a plus-shaped BUW epidermal interface was designed and constructed as a plus-shaped touch panel to program a virtual tank movement (Fig. 5 a). Four working segments of the plus-shaped BUW epidermal interface corresponded to four movement modes of the virtual tank, including going forward, moving backward, turning left, and turning right (the left image in Fig. 5 a). Furthermore, to simulate the aiming manipulation of the gun barrel like a practical military exercise, a hexagon-shaped BUW epidermal interface was also designed and constructed to manipulate the gun barrel actions of the virtual tank (Fig. 5 b). The manipulations of the gun barrel were controlled by the hexagon-shaped BUW epidermal interface, including aiming, turning left, turning right, moving upward, moving down, and firing (the left image in Fig. 5 b). In the HMI of virtual tank movement, touching different working segments of the plus-shaped BUW epidermal interface caused the change in the response resistance so that controlling the corresponding virtual tank movement (Fig. 5 c). By touching the specified working segments of the hexagon-shaped BUW epidermal interface, the response resistance would be generated quickly. In this way, the corresponding command would be issued to control the gun barrel actions (Fig. 5 d). It could be found that these response resistances were significantly different regardless of the shape of the BUW epidermal interface, so the commands could be accurately sent according to the response resistances. More specifically, a demonstration of the typical virtual tank movement by the plus-shaped BUW epidermal interface was designed to be a training program for mastering the terrain during a specific military exercise (Fig. 5 e). The primary demonstration of the typical virtual gun barrel by the hexagon-shaped BUW epidermal interface was conducted for practicing the aim and fire of the gun barrel (Fig. 5 f). A vivid control of a virtual tank movement and the gun barrel actions by the BUW epidermal interface with different shapes was displayed in Movie S3, which was designed as a combat scheme of tank control to monitor terrain conditions and bombard the enemy. Due to the well consistency between the real touch sensing and the virtual object operations, the effectiveness of the actual training scenario program under complex human–machine interaction tasks could be greatly improved by the virtual practice. Fig. 5 BUW epidermal interface with different shapes for controlling virtual tank movements and gun barrel actions. a Illustrations of four motion azimuth tracks in controlling tank movement and the plus-shaped BUW epidermal interface. b Illustrations of six operations in controlling the gun barrel and the hexagon-shaped BUW epidermal interface. c Response resistances of the plus-shaped BUW epidermal interface for virtual tank movements. d Response resistances of the hexagon-shaped BUW epidermal interface for gun barrel actions. e , f Typical photos of different processes of virtual tank movement using the plus-shaped BUW epidermal interface and gun barrel actions using the hexagon-shaped BUW epidermal interface"
} | 11,434 |
35294232 | PMC8926335 | pmc | 4,260 | {
"abstract": "Elastic stretchability and function density represent two key figures of merits for stretchable inorganic electronics. Various design strategies have been reported to provide both high levels of stretchability and function density, but the function densities are mostly below 80%. While the stacked device layout can overcome this limitation, the soft elastomers used in previous studies could highly restrict the deformation of stretchable interconnects. Here, we introduce stacked multilayer network materials as a general platform to incorporate individual components and stretchable interconnects, without posing any essential constraint to their deformations. Quantitative analyses show a substantial enhancement (e.g., by ~7.5 times) of elastic stretchability of serpentine interconnects as compared to that based on stacked soft elastomers. The proposed strategy allows demonstration of a miniaturized electronic system (11 mm by 10 mm), with a moderate elastic stretchability (~20%) and an unprecedented areal coverage (~110%), which can serve as compass display, somatosensory mouse, and physiological-signal monitor.",
"introduction": "INTRODUCTION Stretchable electronics represent an area of focusing interest in the past decade, in part owing to the broad spectrum of applications, spreading from health monitoring ( 1 – 8 ) and disease treatment ( 9 – 16 ), to internet of things ( 17 – 20 ) and soft robots ( 21 – 28 ), and to virtual reality and augmented reality ( 29 – 34 ). Stretchable inorganic electronics mainly rely on integration of high-performance inorganic components with elastomer substrates, where ingenious structural designs are key to a high degree of stretchability of the device system, since inorganic electronic components are usually rigid and brittle ( 35 – 42 ). For this class of stretchable electronics, the device stretchability and function density [i.e., the areal coverage ratio of individual components (ICs)] represent two crucial performance metrics of the device system, particularly for miniaturized multifunctional systems, noticing the rapidly growing demand of increased complexity of device functionality ( 43 – 48 ). A higher function density can yield a more miniaturized device system (with a smaller lateral size) than that with a lower function density. While various strategies of stretchable interconnects [e.g., bridge-shaped designs, serpentine interconnects, fractal designs, and helical interconnects ( 49 – 55 )] have been developed, the function densities of device systems based on these technologies (mostly in the form of an island-bridge construction) are typically below 80% because of the fundamental limit (100%) of function density for the single-layer layout. These technologies based on the single-layer layout can hardly achieve, simultaneously, a large function density (e.g., >60%) and a sufficient high stretchability (e.g., >20%) for miniaturized multifunctional systems (e.g., consisting of >15 ICs to realize two or more functions) ( 51 , 56 – 61 ). To bypass the fundamental limit of the single-layer layout, folding-based bilayer layout and stacked multilayer layout were proposed and demonstrated ( 51 , 58 , 62 , 63 ), which have been used to achieve stretchable systems with a function density of ~76%. These bilayer and multilayer devices exploited soft elastomers (with a modulus from several tens of kilopascals to a few hundreds of kilopascals) to encapsulate serpentine interconnects, but such a solid encapsulation strategy could highly restrict the deformation of serpentine interconnects because of their ultrahigh flexibility, thereby posing a limit to the stretchability of the device system. Here, we introduce stacked multilayer network materials (SMNMs) as a general framework for integrating and encapsulating inorganic stretchable electronic devices. While the stacked configuration evidently increases the function density of the system, the cellular encapsulation does not pose any essential constraint to the buckled deformation of stretchable interconnects, thereby enabling a substantial enhancement (e.g., by ~7.5 times) of the elastic stretchability as compared to that with solid encapsulation [e.g., using polydimethylsiloxane (PDMS) with a modulus of 0.81 MPa]. Owing to the high air permeability and biomimetic mechanical properties that match nonlinear stress-strain curves of human skin, these multilayer network materials could enhance the comfortableness when laminated on skin ( 64 – 69 ). Quantitative studies based on finite element analyses (FEA) shed light on the underlying mechanism of constrained deformations of serpentine interconnects encapsulated with network materials and provide rational guidelines for the design optimization. The SMNM-based integration/encapsulation strategy allows the design and demonstration of a millimeter-scale, multifunctional stretchable electronic system (11 mm by 10 mm), with a moderate level (~20%) of elastic stretchability, and, simultaneously, an unprecedented areal coverage (~110%) of ICs. In comparison to previously reported inorganic electronic systems with similar levels of biaxial elastic stretchability and functional complexity ( 51 , 53 , 70 ), the present system offers a much smaller lateral size (by more than twice) and a much higher areal coverage ratio (110% versus 76%). The device system allows high-precision sensing and wireless radio frequency (RF) transmission of temperature, humidity, and 9–degree-of-freedom motion. Demonstrations in compass display, somatosensory mouse, and real-time monitoring of physiological signals suggest a broad range of application opportunities.",
"discussion": "DISCUSSION Collectively, this work presents strategic designs and mechanics analyses that establish SMNMs as a synergistic platform to integrate and encapsulate inorganic stretchable electronic devices. The SMNM-based platform not only offers a significantly enhanced (e.g., by 7.5 times) elastic stretchability of serpentine interconnect by relieving the mechanical constraint but also provides a scalable route to increased function density by exploiting more stacking layers. A highly integrated, miniaturized, stretchable device system with ultrahigh areal coverage (~110%) and small size (11 mm by 10 mm) is demonstrated, with capabilities in compass display, somatosensory mouse control, and physiological signal monitor. The proposed design strategies based on the SMNM have general utilities in various types of electronic devices, especially those (e.g., virtual reality devices and human-machine interfaces) that demand high levels of function density. Development of SMNM-based electronic devices with complex 3D shapes (e.g., tubes and hemispheres) to allow conformal integration with biological organs (e.g., vessels and nerve guide conduits) represents a promising direction for future work."
} | 1,714 |
35654895 | PMC9271586 | pmc | 4,263 | {
"abstract": "Human gut microbial dynamics are highly individualized, making it challenging to link microbiota to health and to design universal microbiome therapies. This individuality is typically attributed to variation in host genetics, diets, environments, and medications, but it could also emerge from fundamental ecological forces that shape microbiota more generally. Here we leverage extensive gut microbial time series from wild baboons—hosts who experience little interindividual dietary and environmental heterogeneity—to test whether gut microbial dynamics are synchronized across hosts or largely idiosyncratic. Despite their shared lifestyles, baboon microbiota were only weakly synchronized. The strongest synchrony occurred among baboons living in the same social group, likely because group members range over the same habitat and simultaneously encounter the same sources of food and water. However, this synchrony was modest compared to each host’s personalized dynamics. In support, host-specific factors, especially host identity, explained, on average, more than 3 times the deviance in longitudinal dynamics compared to factors shared with social group members and 10 times the deviance of factors shared across the host population. These results contribute to mounting evidence that highly idiosyncratic gut microbiomes are not an artifact of modern human environments, and that synchronizing forces in the gut microbiome (e.g., shared environments, diets, and microbial dispersal) are not strong enough to overwhelm key drivers of microbiome personalization, such as host genetics, priority effects, horizontal gene transfer, and functional redundancy.",
"conclusion": "Conclusions We find that gut microbial dynamics are both weakly synchronized across hosts and strongly idiosyncratic to individual hosts. Like members of a poorly coordinated microbial orchestra, microbial communities in different baboons are only weakly “in concert” across the host population. Instead, gut microbial dynamics are idiosyncratic at the level of individual hosts, and each baboon “player” approaches the gut microbial “song” differently. Our results contribute to mounting evidence that forces proposed to synchronize gut microbial metacommunities—shared environments, diets, and between-host microbial dispersal—can create modest synchrony among hosts, especially for hosts living in the same social unit. However, these forces are typically not strong enough to overwhelm powerful and well-known drivers of microbiome personalization, including host genetic effects, individual-level priority effects, horizontal gene transfer, and functional redundancy 16 , 17 , 18 , 19 . Interestingly, these idiosyncratic dynamics were strong even for microbial phyla and families, whose dynamics reflect multiple microbial functions and interactions that potentially buffer them against large fluctuations in abundance. We expect that the personalized dynamics we observed will be even stronger for finer taxonomic levels, especially bacterial species or strains that exhibit a high degree of functional variability across hosts. Understanding if hosts in the same social group or population exhibit shared microbiome dynamics may be useful to researchers interested in predicting individual microbiome changes, linking microbiome dynamics to health outcomes, and designing broadly effective microbiome interventions. These objectives have already been difficult to achieve, in part because of gut microbial personalization in humans and animals. For instance, predictive models of gut microbiome dynamics from one person fail when they are applied to other people 27 . Our results support the idea that microbiome predictions and interventions focused on specific taxa will require personalized approaches. Even then, “universal” microbiome therapies that work the same way for all hosts may be unattainable. Instead, interventions will likely work best when they are designed for host groups or populations that have shared compositions and dynamics. Functional redundancy and horizontal gene flow may also mean that functions will be more predictable than taxa, and prediction and intervention efforts that focus on microbiome functional traits (e.g., metabolite levels; the presence of specific pathways) will likely be less affected by gut microbiome personalization. Together, our results provide novel insights about the extent and ecological causes of microbiome personalization, and they indicate that personalized compositions and dynamics are not an artifact of modern human lifestyles."
} | 1,142 |
30622251 | PMC6325242 | pmc | 4,267 | {
"abstract": "Silver/copper-filament-based resistive switching memory relies on the formation and disruption of a metallic conductive filament (CF) with relatively large surface-to-volume ratio. The nanoscale CF can spontaneously break after formation, with a lifetime ranging from few microseconds to several months, or even years. Controlling and predicting the CF lifetime enables device engineering for a wide range of applications, such as non-volatile memory for data storage, tunable short/long term memory for synaptic neuromorphic computing, and fast selection devices for crosspoint arrays. However, conflictive explanations for the CF retention process are being proposed. Here we show that the CF lifetime can be described by a universal surface-limited self-diffusion mechanism of disruption of the metallic CF. The surface diffusion process provides a new perspective of ion transport mechanism at the nanoscale, explaining the broad range of reported lifetimes, and paving the way for material engineering of resistive switching device for memory and computing applications.",
"introduction": "Introduction Resistive switching (RS) memory, technically referred as resistive random-access memory (RRAM), is a two-terminal device that can change its resistance in response to the electrical stimulus by a voltage pulse, as a result of the formation and disruption of a nanoscale conductive filament (CF) with relatively high conductance due to the high concentration of defects. Defects can originate from the host material, such as oxygen vacancies in a metal oxide based memory 1 , or from the electrodes via ionic migration across the insulating material, e.g., copper (Cu) or silver (Ag) in the so-called conductive bridge random-access memory (CBRAM) 2 . Depending on the specific application, the formed CF has a lifetime, or retention time, ranging from few microseconds to several months, or even years. For example, RS device finds application in non-volatile memories, where the formed CF must have a lifetime in the range of 10 years 1 , 3 . RS device also finds application as a volatile switch which is characterized by a short lifetime in the range of microseconds to milliseconds, providing a feasible technology for fast selection devices in crosspoint arrays of memory or sensor devices 4 , 5 . The tunable lifetime of the CF in the volatile RS device also mimics the short term plasticity of biological synapses 6 , 7 , enabling a burst of novel applications for brain-inspired neuromorphic computing 8 – 11 . Recently, the coexistence of volatile and non-volatile RS within the same device depending on the compliance current during the CF formation has been extensively reported 12 – 14 . However, conflicting explanations are being proposed for the CF lifetimes in non-volatile and volatile RS phenomena. In non-volatile switching, the rupture of metallic filament is usually attributed to the out-diffusion of the metallic atoms from the CF to its host dielectric material 15 , 16 . On the other hand, lifetime in volatile switching is interpreted as the consequence of the atomic clustering to minimize the CF-dielectric interfacial energy 17 – 19 . A comprehensive physical understanding of CF lifetime in volatile and non-volatile RS device may enable a stronger ability to engineer the device materials and operation toward application-based optimization of RS device. Here, we show that metallic CF lifetime is strongly affected by surface atomic self-diffusion. The gradient of surface atomic vacancy concentration induces the migration of atoms on the CF surface toward the minimization of the surface area, leading to clustering of metal atoms and eventually to CF disruption. Theoretical analysis predicts that the surface diffusion effect is only dominant in the sub-10 nm scale at room temperature, due to the high surface-to-volume ratio. The lifetime for disruption of a typical CF can span from microseconds to years for the CF size (diameter) changing from <1 nm size to 14 nm size. The size-dependent lifetime is experimentally validated with a broad range of data for various types of both non-volatile and volatile RS devices. This work provides a new perspective of ion transport mechanism in nanoscale, paving the way for structural and material engineering of nanoionic devices.",
"discussion": "Discussion Surface self-diffusion effect was first observed in vacuum tube electronic devices, as responsible for the blunting of a sub-mm filamentary field-emission cathode operating at elevated temperature (~1000 K) 33 . The lifetime of this type of filament, which controls the vacuum tube lifetime, is generally in the range of hundreds of hours. At nanoscale dimensions, the morphological changes driven by the minimization of surface energy are highly accelerated to the range of observable time scale at room temperature, which is at the origin of a liquid-like pseudoelasticity 31 observed in situ. Surface diffusion effects also control general properties in nanoscale world, such as the lack of sharp tipped metal coated probes for atomic force microscopy 48 compared to the covalent bonded crystallized silicon or diamond probes 49 , and the instability of ultrathin metallic NWs, e.g., Ag NWs with diameter less than 40 nm 36 . In this work, we provide evidence for surface diffusion being the fundamental mechanism for the filament rupture in RRAM devices. Other works have previously proposed that out-diffusion, rather than surface diffusion, acts as the leading dissolution process in other materials systems, such as Ni in NiO 50 . We note, however, that out-diffusion is not expected to play a major role in the materials systems considered in our work, namely Ag or Cu in silk and oxide compounds. This is independently supported by at least two evidences: first, recent TEM observations reveal the presence of Ag or Cu clusters rather than homogeneously distributed Ag within the doped silicon-oxide layer. This was shown for Ag-doped SiO x ( x < 2) 17 , Cu-doped SiO x ( x < 2) 26 , and Ag-doped SiO 2 27 , 34 . All these results indicate that Ag and Cu have relatively low solubility in silicon oxide, thus resulting in the clustering of Ag or Cu atoms. The low solubility of Ag and Cu in the host materials can be understood as the result of the low reactivity of Ag and Cu elements and the strong chemical stability of Si-O valence bond (see Supplementary Note 7 for extended discussion). Second, experimental data are consistent with Herring’s scaling law \\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}$$\\tau \\sim d_0^4$$\\end{document} τ ~ d 0 4 , while one would expect a dependence \\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}$$\\tau \\sim d_0^2$$\\end{document} τ ~ d 0 2 for out-diffusion as reported by our analysis in the Supplementary Note 7 , in agreement with previous results 50 , 51 . Out-diffusion might be non-negligible when the metal has a relatively large solubility in the host materials, such as Ag in Ag 2 S 52 and Cu in CuS 53 . The volatile switching in RS devices has previously been interpreted as the consequence of the atomic clustering to minimize the Gibbs-Thomson energy at the interface between the filament and the host material 17 – 19 , 54 . Our interpretation of filament shape evolution induced by surface diffusion also has roots in the Gibbs-Thomson effect, given the dependence on surface curvature radii in Eq. ( 1 ). Another common phenomenon induced by Gibbs-Thomson effect is Ostwald ripening 55 – 58 , which has been proposed to control the evolution of the particles obtained by filament fragmentation 17 . According to our model, surface diffusion might control the initial stages of the filament disconnection, which also dictates the filament lifetime according to the Herring’s law. Ostwald ripening instead may be responsible for the post-lifetime evolution of the filament particles. In any case, the driving force for the surface diffusion and Ostwald ripening both can be traced back to the Gibbs-Thomson effect (see Supplementary Note 10 and Supplementary Fig. 12 for more discussion). In RS devices, CFs of various sizes can be electrically formed, resulting in a large range of electrically measurable lifetime. According to our surface diffusion model, the ultimate stability of the CF for non-volatile switching arises from a stable capillary bridge between the top and bottom electrodes. Note that this condition might result in a relatively large CF, which conflicts with other requirements of RS devices for practical applications in non-volatile memories. For instance, RS devices should also be easily erasable at relatively low current, which is necessary to minimize the IR voltage drop across lines in crosspoint arrays. An excessive voltage drop, in fact, would lead to unwanted disturbs to unselected cells. The tradeoff between small operating currents and CF stability requires careful device and materials optimization, toward the minimization of the free energy at the CF surface. Note that experimental data show no tradeoff between endurance and operation current (see Supplementary Note 11 for more discussion). Conversely, minimization of the lifetime in the range of few ns might enable the operation of RS device as fast and efficient selector device for crosspoint arrays 20 , 59 . The fast recovery of the off-state is essential in this case to enable random access within the array, where each memory must appear unselected immediately after access for read or set/reset. Materials engineering should guide the selection of CF materials for selector technology to enhance the surface energy and the related surface diffusion effects 19 . In summary, we propose a universal surface diffusion mechanism for the spontaneous rupture of metallic CFs in filamentary RS devices. Surface diffusion consistently accounts for the transition from volatile to non-volatile switching observed in nanoscale RS devices, where the lifetime of sub-10 nm CFs can span from microseconds to years. Results provide a general framework for understanding the stability of nanoscale structures, and designing RS devices for a wide range of applications, e.g., non-volatile memories with high stability for digital data storage, volatile switching for selector devices in crosspoint arrays, and tunable-lifetime RS synapses for neuromorphic computing with short/long-term plasticity."
} | 2,704 |
27342774 | PMC4919853 | pmc | 4,268 | {
"abstract": "Background Succinate is a kind of industrially important C4 platform chemical for synthesis of high value added products. Due to the economical and environmental advantages, considerable efforts on metabolic engineering and synthetic biology have been invested for bio-based production of succinate. Precursor phosphoenolpyruvate (PEP) is consumed for transport and phosphorylation of glucose, and large amounts of byproducts are produced, which are the crucial obstacles preventing the improvement of succinate production. In this study, instead of deleting genes involved in the formation of lactate, acetate and formate, we optimized the central carbon metabolism by targeting at metabolic node PEP to improve succinate production and decrease accumulation of byproducts in engineered E. coli . Results By deleting ptsG , ppc , pykA , maeA and maeB , we constructed the initial succinate-producing strain to achieve succinate yield of 0.22 mol/mol glucose, which was 2.1-fold higher than that of the parent strain. Then, by targeting at both reductive TCA arm and PEP carboxylation, we deleted sdh and co-overexpressed pck and ecaA, which led to a significant improvement in succinate yield of 1.13 mol/mol glucose. After fine-tuning of pykF expression by anti- pykF sRNA, yields of lactate and acetate were decreased by 43.48 and 38.09 %, respectively. The anaerobic stoichiometric model on metabolic network showed that the carbon fraction to succinate of engineered strains was significantly increased at the expense of decreased fluxes to lactate and acetate. In batch fermentation, the optimized strain BKS15 produced succinate with specific productivity of 5.89 mmol gDCW −1 h −1 . Conclusions This report successfully optimizes succinate production by targeting at PEP of the central carbon metabolism. Co-overexpressing pck - ecaA, deleting sdh and finely tuning pykF expression are efficient strategies for improving succinate production and minimizing accumulation of lactate and acetate in metabolically engineered E. coli . Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0284-7) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion In this paper, PEP was selected as optimized target for increased succinate production and attenuated accumulation of byproducts in engineered E. coli under anaerobic conditions. By deleting ptsG , pykA , ppc and maeAB genes, we have designed and constructed initial succinate-producing E. coli strain. The succinate metabolic pathway was then enhanced with deletion of sdh and co-overexpression of pck - ecaA, resulting in succinate production of 25.51 mM. By introducing artificial sRNA of anti- pykF , the titer of succinate in the final optimized strain BKS15 was 30.12 mM with remarkable decrease in lactate and acetate. Metabolic flux analysis and fermentation kinetics showed that our optimization strategy could efficiently enhance the central carbon flux to succinate and decrease to byproducts. Recently, the progress in metabolic engineering suggested that limitation of cellular ATP supply and redox unbalance can be alleviated for improving succinate production in E. coli [ 41 ]. Combination of our strategies with those targets would further develop high succinate-producing microorganisms.",
"discussion": "Results and discussion Initial construction for succinate production The wildtype E. coli BW25113 (DE3) produced a small amount of succinate in the acid mixture (Fig. 2 ) from glucose under anaerobic fermentation conditions, which was consistent with the previous report [ 6 ]. Glucose uptake through PTS system consumes almost half of the available PEP that is the precursor of succinate, which leads to the significantly decreased amounts of PEP for succinate production. In E. coli , the inactivation and mutation of genes involved in the PTS system was beneficial for succinate production [ 25 , 26 ]. Thus, to save PEP from consumption of PTS system, we deleted ptsG gene in strain BW25113 (DE3) and constructed strain BKS4. Succinate production of strain BKS4 was significantly increased with 2.0-fold higher yield than that of strain BW25113 (DE3) ( p < 0.01) (Fig. 2 ). Meanwhile, the yields of lactate and acetate in strain BKS4 were decreased by 17.65 % ( p < 0.05) and 19.83 % ( p < 0.01), respectively. The results indicated that the inactivation of PTS system played an essential role in the availability of PEP to support succinate production. Fig. 2 Yields of succinate, lactate and acetate of initial succinate-producing strains. Error bars represent SD for three replicates. Asterisks indicate p -values (** p < 0.01, * p < 0.05) compared to BW25113 (DE3) In succinate metabolic pathway, the carboxylation of PEP catalyzed by PPC or PCK is a rate-limiting step committed to succinate production. ATP is essentially consumed for PPC catalyzing the formation of OAA from PEP [ 27 ]. On the contrary, one molecule ATP is generated from carboxylation of one molecule PEP catalyzed by PCK. The deletion of pck gene in E. coli remarkably inhibited succinate production as well as the cell growth [ 27 ], indicating that PCK might be more efficient than PPC. In addition, the function of PCK was partially inhibited by PPC under anaerobic fermentation [ 13 , 14 ]. Thus, we deleted ppc gene to enhance energy supplement and activate PCK. Furthermore, both PEP and malate would convert to pyruvate, which is smoothly turned into byproducts lactate, acetate and formate via the decarboxylation, dehydrogenation, and pyruvate-formate lyase, respectively. Formate is further split into carbon dioxide and water by formate dehydrogenase, while lactate and acetate accumulate in fermentation broth. Since the substrate specificity of malic enzymes for malate is 6-fold higher than that for pyruvate, malic enzymes encoded by maeA and maeB tend to catalyze the decarboxylation of malate to pyruvate [ 28 ]. The formation of pyruvate and its derivative byproducts strongly compete with succinate production for PEP and malate. Inactivation of pykA and pykF has been shown to be effective in inhibiting the conversion of PEP to pyruvate [ 29 ]. Consequently, in order to inhibit the formation of pyruvate from PEP and malate, we deleted pykA , maeA and maeB genes. Unfortunately, compared to strain BKS4, strain BKS8 with deletion of pykA , ppc , maeA and maeB did neither significantly attenuate the accumulation of lactate and acetate, nor increase the succinate yield (Fig. 2 ). The low expression level of pck gene in wild-type E. coli could result in the insufficient metabolic flux to OAA [ 27 ], and pykF might be more active than pykA in the formation of pyruvate from PEP. It suggested that pck and pykF genes could be the potential targets. Therefore, using initial strain BKS8, we further optimize these two targets of succinate metabolic pathway to improve succinate production. Combined optimization of targeting at TCA cycle and carboxylation of PEP to increase succinate production Succinate, an essential intermediate of TCA cycle, cannot be efficiently accumulated in E. coli fermentation. In order to increase succinate production, we optimized succinate metabolic pathway by preventing the backflow of succinate to fumarate, activating glyoxylate shunt bypass to decrease the requirement of reducing power, and co-overexpressing pck - ecaA to fix CO 2 more efficiently. Succinate dehydrogenase (SHD) encoded by sdh gene catalyzes the dehydrogenation of succinate to fumarate. The sdh expression was not totally inhibited under anaerobic conditions [ 30 ]. Herein, we deleted sdh gene to enhance the reductive TCA arm and block the conversion of succinate to fumarate in strain BKS8 background. As expected, the titer and yield of succinate in strain BKS9 were increased by 55.24 % (7.11 mM) ( p < 0.05) and 50.00 % (0.33 mol/mol glucose) ( p < 0.05), respectively (Fig. 3 ). The inactivation of sdh gene showed to increase succinate production in E. coli and Corynebacterium glutamicum under aerobic conditions [ 31 – 33 ]. To the best of our knowledge, sdh gene was first deleted to improve anaerobic succinate production in our study. Fig. 3 Deletion of sdh and iclR , and co-overexpression of pck - ecaA increased succinate production. Error bars represent SD for three replicates. Asterisks indicate p -values (** p < 0.01, * p < 0.05) in which BKS9 and BKS10 were compared to BKS8 and BKS11 was compared to BKS10 Glyoxylate shunt bypass could recover the metabolic flux of the oxidative TCA arm and acetyl-CoA of pyruvate metabolism with less reducing power used, and might contribute to succinate production. The aceBAK operon coding isocitrate lyase, malate synthase and isocitrate dehydrogenase kinase is responsible for the glyoxylate shunt bypass. The transcription of the aceBAK operon is tightly repressed by transcription factor IclR, but induced by inactivating iclR gene [ 34 ]. Thus, the deletion of iclR gene resulted in strain BKS10. As shown in Fig. 3 , the titer and yield of succinate in strain BKS10 was not apparently increased. It was likely that the gene expression involved in glyoxylate bypass are complex and regulated by multiple factors [ 35 ] and deletion of iclR was not sufficient for activating glyoxylate shunt bypass [ 36 ]. Conversion of PEP to OAA in succinate metabolic pathway is net carbon integrated via CO 2 fixation catalyzed by PCK. In fact, the active substrate for PCK is not CO 2 , but the chemically less reactive bicarbonate anion (HCO 3 − ) [ 37 ]. Thus, CaCO 3 , MgCO 3 or NaHCO 3 were often added to the culture media. CO 2 is more permeable across cell membrane than HCO 3 − , but the hydration reaction rate of CO 2 to HCO 3 − is relatively slow. There might not be enough HCO 3 − spontaneously made in vivo to access succinate production. Carbonic anhydrase encoded by ecaA gene catalyzes the hydration of intracellular CO 2 to HCO 3 − . Expression of ecaA gene of cyanobacterium Anabaena in E. coli led to an obvious increase in succinate production [ 38 , 39 ]. Thus, the ecaA gene was co-expressed with pck in strain BKS10, generating strain BKS11. Compared to strain BKS10, combinatorial expression of pck - ecaA in strain BKS11 resulted in a 2.2-fold increase in succinate yield (1.16 mol/mol glucose) ( p < 0.01) and a 1.2-fold increase in succinate titer (18.17 mM) ( p < 0.01) (Fig. 3 ). Fine tuning of pykF expression to improve succinate production Although succinate production was increased remarkably in engineered strains, the yields and titers of lactate and acetate remained high by using the strategies aforementioned in the text (Fig. 4b, c, d ), which suggested that metabolic flux from PEP to pyruvate was relatively strong. Deletion of maeA and maeB and pykA did not significantly attenuated the accumulation of lactate and acetate (Fig. 2 ), suggesting that pykF gene might dominate the formation of pyruvate. Thus the strategy of synthetic small RNA (sRNA) engineering [ 40 ] was used to finely tune the expression of pykF to attenuate the accumulation of lactate and acetate. Fig. 4 Fine tuning of pykF expression strength to improve succinate production and attenuate accumulation of lactate and acetate. a Two anti- pykF sRNA plasmids were designed and constructed at different expression levels by combinations of promoters and plasmid copy number. (H) and (L) represented high-copy-number plasmid (pRSF) and low-copy-number plasmid (pBldgbrick2), respectively. b Relative yields of succinate, lactate and acetate. BKS12 was compared to BKS8 and BKS13 and BKS14 were compared to BKS12. c Yields of succinate, lactate and acetate. The significance was compared to BKS11. d Titers of succinate, lactate and acetate. The significance was compared to BKS11. Error bars represent SD for three replicates. Asterisks indicate p -values (** p < 0.01, * p < 0.05) Using AUG to nucleotide +24 of the pykF mRNA as the binding sequence and selecting E. coli micC as the scaffold, anti- pykF sRNA working sequence was designed (Fig. 4a ). We used two kinds of plasmids with different copy number and tested the inhibitory effects of anti- pykF sRNA on the accumulation of lactate and acetate in strain BKS12 with overexpression of pck gene. When anti- pykF sRNA was expressed on the high-copy-number plasmid pRSF and under the control of T7 promoter, no obvious changes were observed in the yields of succinate, lactate and acetate (Fig. 4b ). Then, we constructed the low-copy-number plasmid pBldg-anti-pykF with a pY15A origin of replication, and expression of anti- pykF was controlled under lacUV5 promoter. The metabolite analysis of engineered strain BKS14 showed that the yields of lactate and acetate were decreased by 55.77 % ( p < 0.01) and 47.73 % ( p < 0.01), respectively, and the yield of succinate was increased by 23.38 % ( p < 0.05) compared to BKS12(Fig 4b ). We further tested whether the expression of anti- pykF under the control of lacUV5 promoter in strain BKS11 would improve succinate production and attenuate accumulation of byproducts. pBldg-anti-pykF was transformed into strain BKS11, generating strain BKS15. Compared to strain BKS11, the low expression of anti- pykF in strain BKS15 led to the decrease of 43.48 % ( p < 0.05) and 38.09 % ( p < 0.01) in the yields of lactate and acetate, respectively (Fig 4c ). Although succinate yield of strain BKS15 was not improved, succinate titer was increased by 13.43 % ( p < 0.05) (Fig. 4d ). The results showed that the down-regulated formation of pyruvate by expressing anti- pykF would enhance the metabolic flux from PEP to succinate. Distribution of intracellular metabolic flux Genetic and metabolic modification used in this study remarkably increased succinate production and attenuated the accumulation of lactate and acetate. However, the intracellular metabolic flux distribution of the metabolic network was still unclear. In order to demonstrate in detail how previous efforts changed the metabolic flux directions and optimized the performance of succinate-producing strains step by step, global metabolic flux analysis was made. The simplified metabolic model that described the metabolic relationship in anaerobically fermentative E. coli was shown in Fig. 5 . This model was comprised of fifteen intermediates and sixteen metabolic reactions designated by V1-V16 (Additional file 1 : Table S1). Among these sixteen reactions, the measurable quantities V1, V6, V16 and (V7 + V10) were used to calculate the metabolic fluxes of other intermediates according to relationships shown in Additional file 1 : Table S2. The estimated metabolic fluxes in mM gDCW −1 h −1 of E. coli stains BW25113(DE3), BKS8, BKS9, BKS10, BKS11 and BKS15 under anaerobic fermentation were presented in Additional file 1 : Table S3. Fig. 5 Metabolic flux analysis of succinate-producing strains. The fluxes in mM gDCW −1 h −1 were calculated according to fermentation data at 40 h and normalized by glucose consumption rate as well as expressed in a basis of 100 As shown in Fig. 5 , metabolic modifications led to the fact that fluxes to OAA (V5), malate (V12), fumarate (V13), succinate (V15 and V16) were significantly increased and that fluxes to pyruvate (V4), lactate (V6), and acetate (V7 + V10) were remarkably decreased from strains BW25113(DE3) to BKS15. The results indicated that our strategies favored the improvement of succinate production and the decrease of byproduct accumulation. The split ratios of fluxes to OAA, PYR, lactate, acetate and succinate were obtained by analyzing the PEP, PYR, acetyl-CoA and succinate nodes. As shown in Table 1 , compared to strain BW25113 (DE3), the fraction of the metabolic flux diverted to OAA from PEP node (V5/V3) in strain BKS8 increased by 1.8-fold ( p < 0.01), corresponding 2.2-fold fraction increase of the metabolic flux to succinate (V16/V3) ( p < 0.01). Pentuple deletions of ptsG , ppc , pykA , maeA and maeB could significantly streamline PEP pool for succinate production. Strain BKS9 showed the increase of the metabolic flux to succinate (V16/V3), indicating the deletion of sdh gene resulted in more metabolic flux to OAA from PEP node (V5/V3). Strain BKS10 did not show carbon flux through glyoxylate shunt bypass (V11 = 0) in the stoichiometric model, indicating that deletion of iclR gene did not activate glyoxylate shunt bypass. Table 1 Split ratios of fluxes to OAA, PYR, lactate, acetate and succinate Strains Fraction of PEP to OAA (V5/V3) Fraction of PYR production (V4/V3) Fraction of lactate production ( V6/V3) Fraction of acetate production (V7 + V10)/V3 Fraction of succinate production (V16/V3) BW25113(DE3) 3.08 ± 0.02 % 96.92 ± 0.02 % 23.46 ± 0.71 % 77.25 ± 2.66 % 2.61 ± 0.02 % BKS8 8.53 ± 0.03 % 91.47 ± 0.02 % 19.43 ± 0.48 % 73.70 ± 1.56 % 8.29 ± 0.01 % BKS9 12.39 ± 0.45 % 86.93 ± 1.43 % 19.95 ± 0.14 % 66.74 ± 0.08 % 12.61 ± 0.47 % BKS10 13.66 ± 0.22 % 86.34 ± 1.10 % 18.28 ± 0.44 % 67.84 ± 2.89 % 13.66 ± 0.22 % BKS11 45.94 ± 0.73 % 53.87 ± 0.72 % 16.97 ± 0.59 % 36.90 ± 0.32 % 55.54 ± 0.98 % BKS15 52.31 ± 0.83 % 47.69 ± 0.67 % 14.88 ± 0.76 % 29.91 ± 0.70 % 67.20 ± 0.78 % In strain BKS11, 45.94 % of PEP was converted to OAA (V5/V3), 2.4-fold higher than that of strain BKS10 ( p < 0.01). As a result, the fraction of the metabolic flux to succinate (V16/V3) increased from 13.66 % in strain BKS10 to 55.54 % in strain BKS11 (Table 1 ) ( p < 0.01). Meanwhile, strain BKS11 showed lower acetic fluxes ((V7 + V10)/V3). This indicated that co-overexpression of pck - ecaA could significantly enhanced the metabolic flux of PEP to OAA, and simultaneously inhibit other metabolic branches. Compared to strain BKS11, the fractions of the metabolic flux to lactate (V6/V3) and acetate ((V7 + V10)/V3) of strain BKS15 decreased by 12.32 % ( p < 0.05) and 18.94 % ( p < 0.01), respectively (Table 1 ), indicating that expression of anti- pykF attenuated the accumulation of lactate and acetate. At last, with a series of metabolic modifications, compared to strain BW25113(DE3), the final fraction of the metabolic flux to succinate in BKS15 was increased by 24.8 fold ( p < 0.01) and those to lactate and acetate were decreased by 36.57 % ( p < 0.01) and 61.28 % ( p < 0.01), respectively. Anaerobic batch fermentation for succinate production To estimate the fermentation behaviors of engineered succinate-producing strains, anaerobic batch experiments were conducted. The titers, yields , specific productivities and productivities of succinate, lactate and acetate in 70 h fermentation were summarized in Table 2 . As shown in Fig. 6 , the distribution pattern of glucose metabolism and the production of succinate, lactate and acetate were remarkably changed. Strain BKS10 exhausted almost glucose, and accumulated large amounts of lactate and acetate, and a small amount of succinate in 70 h fermentation. Compared to strian BKS10, co-overexpression of pck - ecaA in strain BKS11 retarded glucose consumption, but achieved higher succinate production (25.51 mM), higher succinate yield (0.92 mol/mol glucose) and higher succinate specific productivity (3.96 mmol gDCW −1 h −1 ), increased by 1.9- ( p < 0.01), 1.9- ( p < 0.01) and 2.6-fold ( p < 0.01), respectively. Moreover, the accumulation of lactate and acetate was significantly attenuated. When anti- pykF was further expressed in strain BKS15, glucose was completely consumed and largely distributed to succinate. Production of succinate in strain BKS15 was increased at a linear manner during the fermentation, and the specific productivity of succinate increased by 48.74 % ( p < 0.01); the accumulation of acetate was greatly decreased, and the specific productivity of acetate decreased by 31.64 % ( p < 0.01). Engineered strain BKS15 showed the optimal fermentation performance of higher productivity, titer and yield of succinate with the lower accumulation of lactate and acetate. Table 2 Parameters of succinate production by engineered E. coli strians during anaerobic fermentation Strains Growth rate (h −1 ) Titer (mM) Yield (mol/mol of glucose) Specific productivity (mmol gDCW −1 h −1 ) Productivity (mmol L −1 h −1 ) Succinate Lactate Acetate Succinate Lactate Acetate Succinate Lactate Acetate Succinate Lactate Acetate BKS10 0.071 ± 0.002 8.65 ± 0.73 12.06 ± 0.70 27.13 ± 2.56 0.31 ± 0.02 0.43 ± 0.02 0.98 ± 0.09 1.09 ± 0.06 1.47 ± 0.20 3.31 ± 0.13 0.12 ± 0.01 0.17 ± 0.01 0.39 ± 0.04 BKS11 0.052 ± 0.003 ** \n 25.51 ± 1.79 ** \n 7.82 ± 0.63 ** \n 23.52 ± 1.53 0.92 ± 0.06 ** \n 0.28 ± 0.02 ** \n 0.85 ± 0.05 3.96 ± 0.13 ** \n 1.18 ± 0.04 * \n 3.54 ± 0.08 0.36 ± 0.03 ** \n 0.11 ± 0.01 0.34 ± 0.02 BKS15 0.043 ± 0.002 * \n 30.12 ± 3.31 6.55 ± 0.33 * \n 13.22 ± 1.64 ** \n 1.08 ± 0.11 0.24 ± 0.01 * \n 0.48 ± 0.06 ** \n 5.89 ± 0.41 ** \n 1.20 ± 0.07 2.42 ± 0.19 ** \n 0.43 ± 0.05 0.09 ± 0.01 0.19 ± 0.02 ** \n The data are shown as mean values ± standard deviation (SD) of three replicates. Asterisks indicate p -values (** p < 0.01, * p < 0.05) in which BKS11 was compared to BKS10 and BKS15 was compared to BKS11 Fig. 6 Anaerobic fermentation of engineered succinate-producing strains. a BKS10, b BKS11, c BKS15"
} | 5,356 |
28638368 | PMC5461252 | pmc | 4,270 | {
"abstract": "The performance of microbial electrochemical cells depends upon microbial community structure and metabolic activity of the electrode biofilms. Iron as a signal affects biofilm development and enrichment of exoelectrogenic bacteria. In this study, the effect of ferrous iron on microbial communities of the electrode biofilms in microbial fuel cells (MFCs) was investigated. Voltage production showed that ferrous iron of 100 μM facilitated MFC start-up compared to 150 μM, 200 μM, and without supplement of ferrous iron. However, higher concentration of ferrous iron had an inhibitive influence on current generation after 30 days of operation. Illumina Hiseq sequencing of 16S rRNA gene amplicons indicated that ferrous iron substantially changed microbial community structures of both anode and cathode biofilms. Principal component analysis showed that the response of microbial communities of the anode biofilms to higher concentration of ferrous iron was more sensitive. The majority of predominant populations of the anode biofilms in MFCs belonged to Geobacter , which was different from the populations of the cathode biofilms. An obvious shift of community structures of the cathode biofilms occurred after ferrous iron addition. This study implied that ferrous iron influenced the power output and microbial community of MFCs.",
"introduction": "Introduction Microbial electrochemical cell (MEC) has been admired as a versatile device that can be used for alternative energy generation, electrosynthesis, biosensor, and waste treatment ( Hou et al., 2016 ; Liu et al., 2016a ; Huang et al., 2017 ). However, practical implementation of microbial fuel cells (MFCs) remains restricted by reasons of low electron transfer efficiency and high material costs ( Logan et al., 2006 ). For the past few years, researchers studied electrode materials, exoelectrogenic bacteria, reactor configuration and operational conditions of MFCs ( Watson and Logan, 2010 ; Yong et al., 2011 ; Janicek, 2015 ), and pointed out that microbial biofilm was the most direct and key element that affect current generation ( Mohan et al., 2008 ). However, microbial biofilm and its community structure of MFCs can be influenced by temperature, pH, carbon source, inoculum, and metal ion ( Lu et al., 2011 , 2012 ; Patil et al., 2011 ; Wu et al., 2013 ). The diverse populations developed in the biofilms in MECs have been widely analyzed ( Mei et al., 2015 ). Geobacter as a typical dissimilatory metal-reducing bacterium (DMRB) is commonly identified in MFCs ( Mohan et al., 2014 ; Zhu et al., 2014 ; Kumar et al., 2016 ). Hence, to understand and optimize ecological conditions that facilitate exoelectrogens enrichment and electron transfer are essential for MEC application. Iron plays a central role in the development and maintenance of biofilm of Pseudomonas ( Hunter et al., 2013 ). Although ferric iron has been identified as an important parameter affecting the biofilm formation ( Banin et al., 2005 ), the impact of ferrous iron on the biofilm is less known. Metal ions are essential minerals to composite microorganisms and biological molecules, including metalloproteins which play key roles in most biological processes (iron for respiration; Cvetkovic et al., 2010 ). The reactive metal ions may have the phenomenon of redox reaction, catalysis, or precipitation, etc. and thus directly affect the performance of MECs by influencing the metabolism of microorganisms or the activity of enzymes ( Lu et al., 2015 ). Due to its high redox activity, the Fe 2+ is able to be oxidized at the anode in an air-cathode fuel cells which are capable of abiotic electricity generation ( Cheng et al., 2007 ). The addition of ferrous sulfate to the anode medium has improved the power densities of MFCs during start-up period ( Wei et al., 2013 ). However, there are less literatures concerning the response of exoelectrogenic community in the electrode biofilms to ferrous iron. Ferrous iron used in catholyte of dual-chambered MFC enhanced power output by increasing salt concentration or improving cathode potential ( Ter Heijne et al., 2007 ). A comparison of results with and without ferrous iron as a cathodic reactant also revealed that the addition of ferrous iron enhanced power generation in batch MFC ( Wang et al., 2011 ). However, the knowledge related to the effects of ferrous iron on performances of MFCs and microbial communities of electrode biofilms is less known. To reveal the response of microbial community of the electrode biofilm to ferrous iron, in this study, electrochemical performances of MFCs supplemented with different concentrations of ferrous iron were investigated. Meanwhile, microbial community structures of the anodes and cathodes biofilms in MFCs were analyzed using Illumina Hiseq sequencing of 16S rRNA gene amplicons.",
"discussion": "Results and Discussion Electricity Generation and Electrochemical Activity of MFCs Cyclic voltammetry curves showed that MFCs supplemented with 100 μM ferrous ion (Fe 2+ ) obtained the highest current peak on the 15th day ( Figure 1 ). The results suggested that low concentration of Fe 2+ could obviously improve electrochemical activity of MFCs in the start-up period. During another 15 days of operation, MFCs with 100 μM ferrous ion showed the best electrochemical characteristics compared to MFCs with 150 and 200 μM Fe 2+ , and MFCs without additional Fe 2+ supplement ( Figure 2 ). The maximum voltage of 0.55 V was monitored in MFCs fed with 100 μM Fe 2+ , and then following the order control (0.54 V), 150 μM Fe 2+ (0.52 V) and 200 μM Fe 2+ (0.47 V). After all MFCs were operated for 30 days, MFCs of control groups maintained the steady voltage output, while other MFCs with Fe 2+ addition performed a weaken efficiency. FIGURE 1 Cyclic voltammetry curves of MFCs supplemented with different concentrations of ferrous iron on 15th day . FIGURE 2 Voltage curves of MFCs supplemented with ferrous iron of different concentrations . Community Diversity of MFCs with Different Concentrations of Fe 2+ Since the power outputs of MFCs with 150 and 200 μM were similar, and the CV result of 200 μM adequately represented the decrease of electrochemical activity of electrode biofilms, the biofilm samples of MFCs with ferrous iron of 150 μM were not used for microbial community analysis. After quality filtering the raw tags, 50,373 to 54,932 effective tags were obtained per sample, with average length of 373 bp. Total OTUs at the 97% similarity were ranged from 630 to 824 per sample with an average of 710 OTUs ( Table 1 ). The anode biofilms in MFCs supplemented with ferrous iron showed slightly lower population diversity than that in control MFCs without ferrous iron supplement. Shannon indices were 3.72, 4.71, and 5.21 for the anodes biofilms with 100, 200 μM Fe 2+ , and without Fe 2+ , respectively. By contrast, Fe 2+ increased the population diversities of the cathode biofilms, Shannon indices increased from 4.3 (control) to 5.02 (100 μM Fe 2+ ) and 5.54 (200 μM Fe 2+ ), suggesting that Fe 2+ affected microbial community structure of the electrode biofilms in MFCs. Principal component analysis based on OTUs showed three clusters, the anode biofilms of MFC without Fe 2+ was separated from the anode biofilms of MFC supplemented with Fe 2+ of 100 and 200 μM Fe 2+ and the cathode biofilms (control, 100, and 200 μM Fe 2+ ; Figure 3 ). Table 1 Qualities of reads identified by Illumina Hiseq sequencing and bacterial diversity estimates based on OTUs (97% similarity). Sample name Effective tags OTUs Shannon Chao1 Simpson ACE Good’s coverage Anode (control) 53,807 824 5.21 908.307 0.884 900.018 0.997 Anode (100 μM) 53,136 630 3.716 691.84 0.733 703.657 0.998 Anode (200 μM) 54,932 679 4.706 785.135 0.886 796.327 0.997 Cathode (control) 51,054 692 4.3 755.5 0.748 773.924 0.997 Cathode (100 μM) 54,592 697 5.021 757.026 0.879 771.527 0.998 Cathode (200 μM) 50,373 741 5.542 810.327 0.927 813.045 0.997 FIGURE 3 Principal component analysis based on operational taxonomic units of the anode and cathode biofilms of MFCs . Bacterial Composition of the Anode and Cathode Biofilms The bacterial communities of the anode biofilms were substantially shifted when additional Fe 2+ was supplemented in MFCs. Proteobacteria were the most dominant phylum observed both in the anode (71–75%, relative abundance) and cathode biofilms (41–78%) ( Figure 4A ). Chlorobi (11–14%) and Bacteroidetes (4–8%) were also predominant phyla in the anode biofilms. The relative abundances of Lentisphaerae in the cathode biofilms, were much higher than that in the anode biofilms, reached to 31% (100 μM Fe 2+ ), 22% (200 μM Fe 2+ ), and 4% (control). Deltaproteobacteria , Ignavibacteria , and Betaproteobacteria were the most predominant classes in the anode biofilms and accounted for 75% more or less, of which, the abundance of Deltaproteobacteria in the anode of MFCs with 100 μM reached to 50%, speculating that Deltaproteobacteria were the dominant class since MFC start-up period ( Figure 4B ). By contrast, microbial community structures of cathodes were different from anodes. Alphaproteobacteria , Gammaproteobacteria , Bacteroidia , and Lentisphaeria were the predominant classes on the cathodes. Cathodes of MFCs with additional Fe 2+ had similar communities that were much different with control group. FIGURE 4 Microbial community taxonomic wind-rose plots based on relative abundance of 16S rRNA sequences of the anode and cathode biofilms in MFCs at the phylum \n (A) and class levels (B) . The predominant genera varied significantly among all anodes and cathodes biofilms ( Figure 5 ). The majority of predominant populations in the control MFCs were affiliated with Geobacter spp. (30.7%) and Legionella spp. (50.3%). Geobacter was also the predominant genus in the anode of MFC supplemented with 100 and 200 μM Fe 2+ , the relative abundance of which population reached up to 49.3 and 24.4%. Another predominant genus in the anode biofilms of MFC (200 μM Fe 2+ ) was affiliated to Rhodanobacter (19%). In the cathode biofilms of MFCs with 100 and 200 μM Fe 2+ , higher relative abundance of predominant genera belonged to Legionella spp. (2 and 6%), and no absolutely predominant populations were present. Hierarchical cluster analysis of microbial communities based on genus taxonomy revealed that the relative abundance of Sphaerochaeta , Dechloromonas , Paracoccus , Thermomonas , and Rhodanobacter increased in the anode biofilms of MFCs supplemented with 200 μM Fe 2+ ( Figure 6 ). Meanwhile, the relative abundance of Gordonia , Sphingopyxis , Hydrogenophaga , and Janthinobacterium in the cathode biofilms of MFCs with 200 μM Fe 2+ were relatively higher than that in the cathodes biofilms of MFCs without Fe 2+ and with 100 μM Fe 2+ , but higher proportion of Thauera , Dokdonella , Fusibacter , Devosia , and Desulfovibrio were observed in the cathode biofilms of MFCs with 100 μM Fe 2+ . FIGURE 5 Relative abundance of predominant genera in the anode and cathode biofilms in MFCs supplemented with different concentrations of ferrous iron . FIGURE 6 Hierarchical cluster analysis of predominant populations in the anode and cathode biofilms in MFCs. The genera with the relative abundance of the top 35 are shown. The species clustering tree is on the left and the sample clustering tree is on the top. Each box of the heatmap represents a Z -score, a positive score indicates a datum above the mean, while a negative score indicates a datum below the mean. Effect of Fe 2+ on Predominant Populations in the Electrode Biofilms Ferrous iron with appropriate concentration (100 μM) stimulated electrochemical activity of MFCs during the start-up period, but Fe 2+ cannot enhance power output after 30 days of operation and higher concentration of Fe 2+ had the negative effect ( Wei et al., 2013 ), presumably the Fe 2+ facilitated biofilm formation at the early stage. The metal ions may act as redox active sites in the enzymes which catalyze the electron transfer and redox reaction to affect the performance of bio-electrochemical systems (BESs) ( Lu et al., 2015 ). In mature anode biofilms, pH decreased through different growth phases, showing that the pH is not always a limiting factor in a biofilm. Meanwhile, increasing redox potential at the biofilm electrode was associated only with the biofilm, demonstrating that microbial biofilms respire in a unique internal environment ( Babauta et al., 2012 ). Oxidation of ferrous ion by microbes is an important component of iron geochemical cycle ( Croal et al., 2004 ). Recent studies also confirmed that Fe 2+ oxidation provides an energetic benefit for some microbes’ growth when using Fe 2+ and acetate as the co-substrate ( Muehe et al., 2009 ; Chakraborty et al., 2011 ). Illumina Hiseq sequencing of 16S rRNA gene indicated that Fe 2+ shifted bacterial community and influenced enrichment of exoelectrogenic bacteria in the anode biofilms. An excessive amount of metal salts may result in negative effects on the performance of BESs by inhibiting the activity of microorganisms ( Jiang et al., 2011 ). The relative abundance of Geobacter increased from 30.7 to 49.3% in MFCs with 100 μM Fe 2+ but decreased to 24.4% in MFCs with 200 μM Fe 2+ , implying higher Fe 2+ concentration could not further enrich Geobacter . As a result, the power output of MFC with higher Fe 2+ concentration (200 μM) was lower than control and 100 μM Fe 2+ during MFC steady operation. Rhodanobacter accounted for a large proportion (19%) in MFCs with Fe 2+ concentration of 200 μM. To date, the function of Rhodanobacter was mostly investigated on denitrifying ( Green et al., 2012 ) and thiosulfate-oxidizing ( Lee et al., 2007 ), but little is reported about Fe 2+ oxidation especially mediated by C-type cytochromes ( Croal et al., 2007 ; Bird et al., 2011 ). Whether it participates in interspecies interaction with Geobacter should be further proved. Other exoelectrogenic bacteria also formed a certain proportion in different anode biofilms, such as Pseudomonas (1–6%) and Arcobacter (3–7%) ( Fedorovich et al., 2009 ; Yong et al., 2011 ). Pseudomonas has a positive role to benefit other exoelectrogens in anode biofilm under a high concentration of salt addition ( Liu et al., 2016b ). Arcobacter can be selectively enriched in an acetate-fed MFC and rapidly generates a strong electronegative potential ( Fedorovich et al., 2009 ). It indicated that additional ions, like Fe 2+ , will take part in biofilm metabolism or microbial communication, which resulted in community structure changes. The microbial communities on the cathodes clearly differed from the anodes biofilms in all MFCs. The most predominant genera in the cathode biofilms of MFCs without additional ferrous iron came from Legionella spp. (50.3% of relative abundance). However, the relative abundance of Legionella on the cathode biofilms declined to 2–6% with Fe 2+ addition, suggesting that Legionella was inhibited by high concentration of Fe 2+ . The abundance of Fe(II)-oxidizing bacteria, Janthinobacterium ( Geissler et al., 2011 ), in the cathode biofilms of MFC with 200 μM Fe 2+ were relatively higher than other groups ( Figure 6 ). Hierarchical cluster analysis based on genus taxonomy demonstrated that the response of predominant populations in the electrode biofilms to ferrous iron occurred, indicating the effect of ferrous iron on microbial community in MFCs. Effect of Environmental Factors on MFC Performances Some environmental factors, such as nutrients, pH, and temperature, influence the performances of MFCs by changing microbial activity and community structure. Our study indicated that ferrous iron changed microbial community structures of electrode biofilms of MFCs. Other metals (e.g., Ca, Mg, Pt, Au, Pd, Fe, V, Mn) and metal-nanomaterials affected current generation of MECs by changing the metabolism and enzyme activity of microorganisms ( Lu et al., 2015 ). These studies have analyzed effect of single metal on electricity generation by MFCs, however, the effect of combined metals on microbial community structure and performance of MFCs should be further investigated. Neutral pH is considered as the optimal condition for current generation by MFCs ( Gil et al., 2003 ; Jadhav and Ghangrekar, 2009 ). However, a higher pH has been demonstrated to enhance the electrochemical activity of riboflavin which is a metabolite responsible for extracellular electron transfer in some species ( Yuan et al., 2011 ; Yong et al., 2013 ). By contrast, MFCs have also been operated at pH less than 4.0 and produced high current densities by acidophilic bacterium ( Malki et al., 2008 ; Winfield et al., 2016 ). Previous studies proved that temperate substantially affected the performances of MECs or MFCs by shaping microbial community ( Lu et al., 2011 , 2012 ). Synergistic effect of metals, pH and temperature on performances of MECs and correlation analysis of these environmental factors should be further investigated in the future."
} | 4,295 |
25382931 | null | s2 | 4,271 | {
"abstract": "We study the effects of noise on the dynamics of a system of coupled self-propelling particles in the case where the coupling is time-delayed, and the delays are discrete and randomly generated. Previous work has demonstrated that the stability of a class of emerging patterns depends upon all moments of the time delay distribution, and predicts their bifurcation parameter ranges. Near the bifurcations of these patterns, noise may induce a transition from one type of pattern to another. We study the onset of these noise-induced swarm re-organizations by numerically simulating the system over a range of noise intensities and for various distributions of the delays. Interestingly, there is a critical noise threshold above which the system is forced to transition from a less organized state to a more organized one. We explore this phenomenon by quantifying this critical noise threshold, and note that transition time between states varies as a function of both the noise intensity and delay distribution."
} | 253 |
28126934 | PMC5270693 | pmc | 4,273 | {
"abstract": "ABSTRACT Here, we report the first draft genome sequence (42.38 Mb containing 13,657 genes) of Coniochaeta ligniaria NRRL 30616, an ascomycete with biotechnological relevance in the bioenergy field given its high potential for bioabatement of toxic furanic compounds in plant biomass hydrolysates and its capacity to degrade lignocellulosic material."
} | 88 |
30507067 | PMC7379504 | pmc | 4,274 | {
"abstract": "Summary Microbial populations exist to great depths on Earth, but with apparently insufficient energy supply. Earthquake rock fracturing produces H 2 from mechanochemical water splitting, however, microbial utilization of this widespread potential energy source has not been directly demonstrated. Here, we show experimentally that mechanochemically generated H 2 from granite can be directly, long‐term, utilized by a CH 4 producing microbial community. This is consistent with CH 4 formation in subsurface rock fracturing in the environment. Our results not only support water splitting H 2 generation as a potential deep biosphere energy source, but as an oxidant must also be produced, they suggest that there is also a respiratory oxidant supply in the subsurface which is independent of photosynthesis. This may explain the widespread distribution of facultative aerobes in subsurface environments. A range of common rocks were shown to produce mechanochemical H 2 , and hence, this process should be widespread in the subsurface, with the potential for considerable mineral fuelled CH 4 production.",
"introduction": "Introduction The majority of prokaryotes on Earth live in the subsurface and are present to depths in excess of 3 km (Parkes et al ., 2014 ). These prokaryotes are far away from photosynthetically derived organic matter and oxygen and are under severe energy limitation (Hoehler and Jorgensen, 2013 ). Therefore, subsurface microorganisms maybe be more reliant on the geosphere for energy supply (Pedersen, 2000 ), including H 2 which has a range of geosphere sources. For example: (i) oxidation of ferrous iron containing minerals, predominantly at elevated temperatures – serpentinization (Holm et al ., 2015 ); (ii) radiolysis of water (Lin et al ., 2005 ); (iii) pyrite formation from FeS and H 2 S (Drobner et al ., 1990 ); and (iv) high temperature conversion of water in minerals into H 2 and peroxy linkages (Freund, 1985 ). Low temperature (~20 °C) basalt weathering/oxidation had been suggested to fuel a H 2 ‐based microbial ecosystem in the Columbia River Basalt Aquifer (Stevens and McKinley, 1995 ). However, this community subsequently was considered to be heterotrophic instead, as little H 2 formation occurred under simulated in situ conditions and also because ferrous iron concentrations would have been limiting (Anderson et al ., 1998 ). Despite this, total H 2 flux in continental rocks has been suggested to be highly significant at 0.36–2.27 x 10 11 mol per year (Lollar et al ., 2014 ), and comparable to the seafloor hydrothermal H 2 fluxes that support spectacular marine ecosystems. This flux would help explain the large terrestrial subsurface biosphere, but H 2 from water radiolysis and serpentinization would be restricted to rocks with radioactive compounds or ferrous iron minerals respectively. Another source of geologically‐generated H 2 is from mechanochemical splitting of water due to free radical reactions on fractured rock surfaces (Kita et al ., 1982 ; Freund et al ., 2002) or rocks under tension (Balk et al ., 2009 ). However, mechanochemical H 2 formation is rarely considered as a deep biosphere energy source despite this process being widespread and not limited to a few specific rock types (Kita et al., 1982; Freund et al ., 2002 ). Although fracturing is concentrated around earthquake zones (Wakita et al ., 1980 ; Brauer et al ., 2005 ), rock comminution during erosion (Telling et al ., 2015 ) and seismic events (Sleep and Zoback, 2007 ), are also sources of mechanochemical H 2 and together these should be widespread in the subsurface. Estimates of mechanochemically produced H 2 at 3.4 × 10 16 mol per year (Hirose et al ., 2011 ; 2012 ) show that it is a larger global H 2 source than serpentinization and water radiolysis combined. In addition, the presence of CH 4 in earthquake zones (Brauer et al ., 2005 ) suggests that some of this mechanically produced H 2 is being used directly by subsurface methanogens. However, it is unknown if the production rates and concentrations of mineral‐H 2 , the conditions for its production (e.g. temperature and pressures) and/or the by‐products of the reactions (e.g. highly reactive oxygen species), would actually enable utilization by anaerobic microbial communities. Investigating whether mechanically‐produced H 2 can be directly utilized by prokaryotic communities is not only important for understanding deep biosphere energy sources, if a significant amount of this H 2 is utilized to form CH 4 , this would also be important for accurate quantification of greenhouse gas formation and global warming. Furthermore, mechanochemical‐H 2 formation may have been important for early life on Earth and could potentially maintain subsurface biospheres on other planets (McMahon et al ., 2016 ). We, therefore, conducted laboratory rock‐crushing experiments under optimal conditions for H 2 ‐utilizing methanogens to test whether mechanochemical‐H 2 formation could directly fuel microbial activity, and hence, potentially microbial ecosystems.",
"discussion": "Results and discussion \n Mineral‐H 2 formation on crushing \n To determine the mineral H 2 formation conditions for subsequent microbial utilization, pure silica (2 g) in vials with aluminium balls under anaerobic conditions were heated at 25, 38, 67, 84 and 100 °C for 30 min and then contents ground using a ball mill (60 min, Supporting Information Fig. S1A ; see Supporting Information for Experimental procedures). The vials were then heated for a further 30 min before headspace gas was analysed. Above ~40 °C H 2 concentrations increased with temperature ( P < 0.05), reaching 178 nmol H 2 L −1 headspace at 100 °C for silica only with milling. All controls, including silica plus water, were not significantly different from an empty vial (Fig. 1 ). These results show that milling and silica were essential for producing significant H 2 , and that other potential sources of H 2 on heating, such as thermal breakdown of organic matter contaminants and rubber stoppers, were negligible sources of H 2 under the prevailing conditions. Furthermore, milling of water with silica produced considerably less H 2 compared with silica without water (Fig. 1 ), suggesting that the added water reduced milling efficiency. This further emphasizes the importance of milling for H 2 formation as does the experiment with silica plus water without milling which produced even less H 2 (~20 nmol L −1 ). Dry grinding of minerals produces H 2 with the water coming from between mineral grains or from reaction of hydroxyl groups (Kameda et al ., 2004 ). Although H 2 formation from silica, and granite, has been shown to increase with temperature, up to a maximum at ~200–220 °C (Kita et al ., 1982 ), lower temperatures are required for direct coupling with microbial H 2 utilization, as the upper temperature for prokaryotes and methanogenesis is around 120 °C (Takai et al ., 2008 ). Hence, there is a compromise between the temperature required for maximum mechanochemical mineral‐H 2 formation, and the temperature range enabling its direct microbial utilization. From the temperature range tested (Fig. 1 ) 67 °C was selected for further experiments to enable subsequent coupling with the deep‐sea, thermophilic methanogen Methanothermococcus okinawensis (growth optimum 60–65 °C, range 40–75 °C, Takai et al ., 2002 ). Prokaryotes at similar thermophilic temperatures have been detected in deep, subsurface sediments (Roussel et al ., 2008 ) and in water from deep rock fracture zones (Takai et al ., 2003 , Moser et al ., 2005 ). Figure 1 Effect of milling on H 2 formation from silica between 25 and 100 °C (mean of triplicates and standard error bars shown). To enhance mineral‐H 2 formation at 67 °C, silica milling was conducted in an oil bath (Supporting Information Fig. S1B ) to provide extended periods of heated milling and this was combined with headspace flushing (Fig. 2 ). Initially with milling, there was rapid H 2 formation decreasing slightly after ~30 h. However, after headspace flushing H 2 rapidly returned to its original concentration, ~490 nmol L −1 . Flushing was repeated another three times up to ~140 h, with the same result, even though milling had stopped after ~55 h. After a further three flushes up to 216 h, the amount of H 2 produced reduced considerably (lowest ~90 nmol L −1 ), indicating that most reactive surfaces had been utilized. However, flushing had resulted in a ~2.5 times increase in the amount of H 2 formed. Another period of milling increased H 2 to above the initial concentration (~760 nmol L −1 ), although subsequent flushing resulted in only low H 2 concentrations (Fig. 2 ). This sequence was repeated in another two milling periods, followed by an extra period without flushing which yielded the maximum H 2 concentrations of 1213 nmol L −1 , after a total of ~530 h. Free radical concentrations also increased with crushing time (Supporting Information Fig. S2 ) corresponding with increasing H 2 formation. These results show that continuous H 2 formation can be obtained by a mixture of (i) additional crushing, and (ii) H 2 removal by headspace flushing. The latter is consistent with feedback inhibition and suggests that microbial H 2 consumption might sustain or even enhance H 2 formation. Cumulative H 2 formation totalled 7186 nmol L −1 . Figure 2 Effect of milling on H 2 from silica at 67 °C. Dotted lines denote headspace flushing (x3 with oxygen free nitrogen). Shading shows milling intervals. Similar results were obtained with crushing basalt with total H 2 production after ~120 h of 350 nmol L −1 , with one initial milling period (~75 h). These results are similar to the initial H 2 formation in low temperature basalt weathering experiments conducted previously by Anderson and colleagues ( 1998 ), who also suggested that initial H 2 formation was possibly due to reactive mineral surfaces, however, our milling was probably more effective, resulting in significant H 2 formation after headspace flushing, which did not occur in the Anderson et al . experiments. Milling at 67 °C for ~30 h also produced H 2 from other minerals in the order of highest concentrations: granite > quartz > silica and borosilicate glass > basalt, ranging in maximum concentration from 1133 to 142 nmol L −1 (Supporting Information Fig. S3 ). Mineralogical changes with crushing granite, including reduction of quartz and formation of new minerals (Supporting Information Fig. S4 , overall decrease in peaks labelled Q for quartz, including some also with other minerals and appearance of additional peaks respectively), confirmed that H 2 generation occurred together with breakage of Si—O bonds in phyllosilicates, which together with free radical formation (Supporting Information Fig. S2 ) is consistent with mechanochemical reactions. \n Coupling mineral derived H 2 with methanogenesis \n For further experiments, the high H 2 producing granite was used in increasing amounts (15–40 g) with 30 g giving maximum H 2 production and then this amount was subsequently used as standard. Under these conditions ~500 μmol L −1 H 2 was produced, but there was no H 2 consumption or CH 4 production when the system was inoculated with a M. okinawensis culture (Supporting Information Fig. S1D ), despite repeated attempts (Supporting Information Fig. S5 ). Under our culture conditions the H 2 threshold for significant CH 4 production by M. okinawensis was between ~200 and 1500 μmol L −1 , so sufficient H 2 was present in our mineral experiments for the methanogen to use. However, mechanochemical splitting of water also produces highly reactive oxygen species (Balk et al ., 2009 ), which could have inhibited this strictly, anaerobic methanogen. Subsurface environments are generally reducing (e.g. H 2 S and reduced metal species), so reactive oxygen species would be reduced, and/or be used directly or indirectly (oxidized products of reduced species ‐ thiosulfate and metal oxides) by facultative aerobes/anaerobes. This would not substantially occur in our pure culture methanogen experiments. Therefore, we specifically enriched a methanogenic community (see Supporting Information Fig. S6 and Experimental Procedures) under low oxygen concentrations (and low H 2, ~400 μmol L −1 ) to inoculate further experiments, which could both cope with oxidized species and produce CH 4 from H 2 (Fig. 4, Supporting Information Fig. S1 ). The same enrichment subculture was then used to inoculate all subsequent experiments (Supporting Information Fig. S6b ), to ensure that the community composition was identical for each. Three experiments were conducted each with a different grinding mechanism to ensure that H 2 formation was not restricted to a specific grinding process. Experiment 1 ‐ rotation with granite balls; Experiment 2 ‐ grinding with a magnetic stirring bar and Experiment 3 ‐ grinding with an abrasive resistant bar to prevent the iron magnet being exposed and contributing to H 2 formation. All experiments resulted in H 2 consumption and CH 4 production after inoculation with the methanogenic community (Fig. 3 ). In Experiments 1 and 2, CH 4 production was almost twice the amount expected from measured H 2 consumption (4H 2 + CO 2 ➔ CH 4 + 2H 2 O), which presumably reflects simultaneous mineral‐H 2 production and H 2 consumption by methanogens. In addition, enhanced H 2 formation similar to the effect of headspace flushing previously documented, and of a similar magnitude (Fig. 2 ), may be occurring due to the methanogenic H 2 consumption. In Experiment 3 (Fig. 3 C), water (4 ml) was added after grinding for 682 h, to further increase H 2 formation (~500 μmol L −1 ) and to demonstrate that the H 2 increase previously observed on addition of the inoculum in Experiments 1 and 2 (Fig. 3 A and B) was due to increased water availability after grinding. After ~1266 h the experiment was inoculated and almost immediately H 2 was consumed and CH 4 produced. By ~250 h incubation most of the H 2 was removed (to ~20 μmol L −1 ) and CH 4 then stabilized around 140 μmol L −1 . Shortly after this, grinding was restarted (after 1587 h) and CH 4 production restarted immediately, but for H 2 there was a delay of ~140 h before concentrations increased, presumably due to its initial consumption for methanogenesis. During this second phase of grinding, CH 4 and H 2 concentrations plateaued at ~160 and 115 μmol L −1 respectively). Grinding was then stopped (2161 h) and H 2 and CH 4 (small decrease) production ceased. After ~60 h grinding was restarted and immediately H 2 was produced, CH 4 concentrations, however, decreased until the system was re‐inoculated (inoculum presumably dried out), after which H 2 again was removed along with CH 4 production, some 2800 h/120 days after the beginning of the experiment. The initial period of methanogenesis was much more rapid in this experiment compared with Experiments 1 and 2 (2–4 times, Fig. 3 ), and the H 2 :CH 4 ratio was as expected for hydrogenotrophic methanogenesis. The second period of CH 4 formation, however, was much slower, presumably reflecting the much lower H 2 concentrations and the H 2 :CH 4 ratio was similar to the Experiments 1 and 2. Probably the large and very rapid initial phase of H 2 consumption in Experiment 3 masked the effect of continuing mineral H 2 formation. In controls, including an inoculated empty crushing bottle, and an autoclaved inoculum, no coupled H 2 removal and CH 4 production occurred (Supporting Information Fig. S7 ). Some CH 4 was released into the inoculated empty bottle control (max 26 μmol L −1 ), but this represented only a fraction of the CH 4 produced in the inoculated mineral H 2 experiments. The controls demonstrate that CH 4 production was not a result of thermal breakdown of cells or organic matter in the inoculum. The rapid response to renewed mineral grinding (Fig. 3 C) also demonstrates how tight and effective this mineral H 2 methanogenesis system is. Figure 3 Granite milling experiments at 67 °C showing H 2 consumption and CH 4 production when inoculated with a methanogenic community.\n Experiment 1: rotating with granite balls. Experiment 2 grinding with magnetic stirrer. Experiment 3: grinding with an abrasive resistant stirring bar. Triangle = H 2 , solid circle = CH 4 . Shading shows milling periods and arrow shows inoculation with methanogenic community. \n \n The methanogenic community \n The composition of the methanogenic community was screened by methanogen functional mcrA gene and 16S rRNA gene sequence analysis (Supporting Information Fig. S8 and Fig. 4 ). Only one methanogenic archaeon was detected in the inoculum and this had 96% ( mcrA gene) and 99% (16S rRNA gene) nucleotide sequence similarity to Methanothermobacter crinale , a methanogen often isolated from subsurface oil and gas reservoirs and thought to develop co‐operative relationships with Bacteria (Cheng et al ., 2011 ). In addition to the methanogen, the methanogenic community also contained several bacterial 16S rRNA gene sequences (Fig. 4 ), predominantly thermophilic Firmicutes , belonging to the orders Thermonanaerobacterales , and Clostridiales , including Desulfotomaculum species, within the Clostridia class. Both of the above bacterial families commonly occur in the deep hot biosphere or deep subsurface environments (Aullo et al ., 2013 ; Parkes et al ., 2014, O'Sullivan et al ., 2015 , Purkamo et al ., 2016 ). An association of methanogens with Clostridiales species have been shown to dominate in deep hot subterranean environments such as deep gold mines (Moser et al ., 2003 ). Many bacterial sequences were related to cultured species (45%), including those from hot springs (Perevalova et al ., 2013 , Brockia lithotrophica , H 2 ‐utilizing, obligate anaerobic, spore‐former), where a H 2 driven methanogenic community has been documented (Chapelle et al ., 2002 ); hot salty environments (Cayol et al ., 1994 , Halothermothrix orenii , an anaerobic, chemoorganotroph); and oil reservoirs (Nilsen et al ., 1996 ), and some have known syntrophic interactions with methanogens (Nilsen et al ., 1996 , Desulfotomaculum thermocisternum, a thermophilic, H 2 ‐utilizing, spore‐forming sulfate‐reducer). In addition, some Desulfotomaculum species have genes encoding for enzymes that can protect against reactive oxygen compounds (Spring et al ., 2009 ). In our system, the presence of Desulfotomaculum species with the methanogen could help methanogenesis to occur despite the presence of oxidized compounds. One of the most common sequences (15%) was related to uncultured Bacteria colonizing young ocean crust (Fig. 4 ), which probably supports significant autotrophic microbial biomass (Bach and Edwards, 2003 ). Comparison of the bacteria in our methanogenic community (see Supporting Information Fig. S9 ) with those detected in H 2 ‐utilizing SLIME environments (Stevens and McKinley, 1995 ) such as aging and young ocean crust (Cowen et al ., 2003 ; Orcutt et al ., 2011 ) or thermophilic methanogenic community fed from cathode‐derived hydrogen (Fu et al ., 2013 ) shows that bacteria in our methanogenic community were representative of populations fuelled by H 2 . Figure 4 Phylogenetic tree of bacterial and archaeal 16S rRNA gene diversity in the methanogenic community inoculum. Neighbour‐joining tree prepared with MEGA 5.2.2 software (method: Jukes‐Cantor model, bootstrap test: 500 replicates) and edited with the Interactive Tree of Life (ITOL) using sequences aligned with the ClustalW2 program. Sequences detected in this study are highlighted in red and bold. \n Summary \n These results demonstrate considerable H 2 formation from a range of common rocks and minerals by crushing induced mechanochemistry. This H 2 formation can be sustained by repeated crushing under anaerobic conditions and at temperatures (25–100 °C) well within the range of prokaryotes. Higher temperatures produce even greater amounts of H 2 (up to a maximum at ~200–220 °C, Kita et al ., 1982 ) and this could diffuse upwards into the temperature limited base of the subsurface biosphere. Granite derived H 2 was directly utilized by a CH 4 ‐producing community in which the methanogen could effectively compete for H 2 (over the 120 days of the experiment), and which was resilient to the oxidized species also mechanochemically produced, but not by a pure methanogen culture on its own. However, this production of oxidized species is also environmentally important as this could help sustain respiratory prokaryotes, independent of oxygen from surface photosynthesis, in the deep subsurface, including those using H 2 or H 2 ‐derived products, such as CH 4 . Subsurface oxidants may also help explain the considerable number of facultative aerobic prokaryotes in the deep subsurface (Pedersen, 1993 ; Miettinen et al ., 2015 ). Estimated earthquake‐derived H 2 flux is five orders of magnitude higher (Hirose et al ., 2012 ) than from radiolysis and serpentinization (Lollar et al ., 2014 ) and, therefore, should be a much more important energy source for the deep biosphere, and provide a continuing fuel for deep CH 4 formation and flux to the atmosphere, which is second only to wetland emissions (Etiope, 2012 ). If all of the estimated flux of mechanochemically derived H 2 (Freund et al ., 2002 ; Hirose et al ., 2012 ) was converted to CH 4 this would be ~1000 times higher than current estimates of geological CH 4 greenhouse gas production (Kirschke et al ., 2013 ). As tectonics on Earth probably occurred by ~3 Ga (Hirose et al ., 2011 ), mechanochemical reaction products would also have been available to early life. Mechanochemistry from landslides, glacial bedrock comminution (Telling et al ., 2015 ) and meteorite impacts add further to this tectonically driven water splitting, rock energy source, which could occur on many other planets (Hurowitz et al ., 2007 ; Yin, 2012 )."
} | 5,566 |
27510763 | null | s2 | 4,275 | {
"abstract": "A biofabrication strategy for creating planar multiscale protein, hydrogel, and cellular patterns, and simultaneously generating microscale topographical features is developed that laterally confines the patterned cells and direct their growth in cell permissive hydrogels."
} | 68 |
40356642 | PMC12066788 | pmc | 4,277 | {
"abstract": "Excessive use and overreliance on chemical fertilizers threatens soil health and environmental sustainability, necessitating eco-friendly alternatives like arbuscular mycorrhizal fungi (AMF). The benefits of AMF are well-documented in staple crops, their effects on diverse species—particularly legumes and non-crop models under uniform conditions—remain underexplored, limiting their scalable adoption. This study evaluated Funneliformis mosseae ’s role in enhancing growth, nutrient uptake, and stress resilience across five species: rice ( Oryza sativa ), sesame ( Sesamum indicum ), sorghum ( Sorghum bicolor ), Egyptian pea ( Sesbania sesban ), and the non-crop Kalanchoe daigremontiana . The pot-experiment was conducted in natural open-field conditions (e.g., ambient light, temperature, and humidity) and inoculated plants were analyzed for biomass yield, nutrient concentrations, and physiological parameters to evaluate F. mosseae ’s efficacy as a sustainable growth promoter. Inoculation with F. mosseae significantly enhanced plant performance across all species. Rice exhibited a 43% increase in dry biomass, alongside 53% higher phosphorus uptake and 24.5% greater magnesium accumulation. Root development improved markedly, with sesame, sorghum, Egyptian pea, and Mexican hat plants showing root length increases of 66.7, 42.9, 35, and 33.3%, respectively. Biomass gains were consistent: Egyptian pea (29% fresh biomass, 33% dry), sesame (30% fresh, 39% dry), sorghum (36.6% total), and Mexican hat plant (31% fresh, 34% dry). Nutrient uptake surged systemically, including potassium (sesame: 42%, Egyptian pea: 17.8%), calcium (sesame: 54.5%, sorghum: 29.4%), and magnesium (Mexican hat plant: 32.4%, Egyptian pea: 22.5%). Physiologically, photosynthetic rates rose by 21.4–45% (highest in Egyptian pea), stomatal conductance improved by 23.3–71.4% (peak in sesame), and chlorophyll a and b levels increased by 30–39.1% and 44.4–150.8%, respectively, across species. These results suggested that F. mosseae could provide a sustainable, environment friendly substitute for chemical fertilizers, preparing for the future of agriculture, where ecological services such as crop productivity and soil fertility depend on mycorrhizas alongside conventional cultivation practices. Integrating AMF into agricultural systems offers a potential strategy for eco-friendly farming practices that are viable and secure for long-term food security and eco-sustainability.",
"conclusion": "5 Conclusion and future perspective Our study provides clear evidence that Funneliformis mosseae inoculation significantly improves plant growth, nutrient uptake, and physiological performance across all tested species. The most notable improvements were seen in rice, which showed a 43% increase in dry biomass, and sesame, which demonstrated a 71.4% improvement in stomatal conductance, might be due to better water uptake and hormonal improvement. We observed particularly strong enhancements in phosphorus uptake in rice (53% increase) and magnesium accumulation in the Mexican hat plant (32.4% increase). Microscopic examination confirmed successful fungal colonization in all treated plants directly linking these benefits to the AMF symbiosis. These results indicate that F. mosseae has the potential to significantly enhance plant growth, nutrient uptake, and physiological performance in a variety of economically and ecologically essential crops. This symbiotic fungus improves biomass yield, nutrient acquisition, and stress resilience and provides a sustainable alternative to chemical fertilizers to remedy problems of soil degradation, environmental pollution, and food security. Future research studies are needed on how plant growth responds when recommended dose fertilizer (RDF) applications are supplemented with Arbuscular Mycorrhizal Fungi inoculation. Research with different fertilizer recommendations will show how well AMF works alongside regular fertilization methods for plant development. Prospective research should be focused on learning how AMF works with fertilizers to help plants grow better without depending more on chemical fertilizers. Future work should focus on long-term field studies to validate its efficacy under diverse agroecological conditions, investigate its interaction with other microbial inoculants, and optimize species-specific applications. Improved integration of F. mosseae into modern agricultural practices is an essential first step towards developing sustainable farming systems that promote healthier soils, minimize environmental footprints, and build resilience to the impact of climate change to guarantee food security and sustainability for the world.",
"introduction": "1 Introduction During the Green Revolution, global crop productivity increased owing to the widespread use of chemical fertilizers, pesticides, and other agricultural inputs. This also allowed the large-scale cultivation of crops and their use to serve a fast-growing population. Despite this, many environmental and agricultural challenges have been caused by overreliance on chemical inputs ( John and Babu, 2021 ). Excessive fertilizers and pesticides have degraded soil health, disturbed ecosystems, and polluted water and air ( Baweja et al., 2020 ). For instance, pesticide usage has doubled worldwide since 1990, with an annual 3.5 million tons ( Sharma et al., 2019 ). However, these practices are not just an assault on biodiversity; they also undermine the long-term fertility of soils, making farmers increasingly dependent on ever-increasing chemical applications to produce yields ( Shattuck et al., 2023 ). As the sustainability of traditional agricultural practices is becoming a concern for researchers and farmers alike, eco-friendly alternative options are being discovered to mitigate these issues ( Gamage et al., 2023 ; Shattuck et al., 2023 ). Plant growth-promoting microorganisms (PGPM) are the most important and have emerged as powerful allies in sustainable agriculture. Bacteria and fungi are microorganisms that enhance nutrient availability in the soil, facilitate plant growth, and decrease environmental stress ( Malgioglio et al., 2022 ; Rehman et al., 2024 ). Arbuscular mycorrhizal fungi (AMF) are vital soil microorganisms that form symbiotic associations with approximately 80% of terrestrial plant species ( Abrar et al., 2024 ). These fungi enhance plant nutrient uptake, primarily phosphorus and nitrogen, by extending their hyphal networks into the soil ( Khan et al., 2024 ). Through this symbiotic relationship, AMF reduce dependence on chemical fertilizers and contribute to sustainable agricultural practices ( Samuel and Veeramani, 2021 ). In turn, the fungi use photosynthates from the host plants as a source of carbon, which, together with polysaccharide-derived carbon, is given as carbon from photosynthates to the fungi, constituting a mutualistic relationship ( Bennett and Groten, 2022 ; Tang et al., 2023 ; Wang et al., 2020 ). Among AMF species, Rhizophagus irregularis and Glomus monosporum are widely recognized for their role in promoting plant growth ( Chandwani et al., 2023 ). However, Funneliformis mosseae has also emerged as a promising AMF species due to its ability to significantly enhance biomass production and nutrient acquisition in various crops. It colonizes plant roots, facilitating better nutrient uptake and optimizing plant physiological performance ( Samuel and Veeramani, 2021 ). F. mosseae also contributes to soil fertility by producing glomalin, a glycoprotein associated with soil aggregation and carbon retention ( Dahiya et al., 2022 ; Agnihotri et al., 2021 ). Despite these advantages, the potential of F. mosseae as a biofertilizer remains underexplored, particularly in diverse crop species. This research study investigated the role of Funneliformis mosseae —a single AMF strain—in enhancing growth, nutrient uptake, and stress resilience across five ecologically diverse plant species: rice ( Oryza sativa ), sesame ( Sesamum indicum ), sorghum ( sorghum bicolor ), Egyptian pea ( Sesbania sesban ), and the non-crop model Mexican hat plant ( Kalanchoe daigremontiana ). Unlike prior studies focusing on individual crops or mixed AMF consortia, our work isolates the specific contributions of F. mosseae under uniform experimental conditions, addressing a critical gap in understanding species-specific AMF interactions. By integrating primary nutrients (N, P, K) with secondary macronutrients (Ca, Mg) and advanced physiological markers (chlorophyll fluorescence, photosynthetic efficiency), this study provides a holistic assessment of AMF-mediated benefits. Furthermore, the inclusion of Mexican hat, a stress-tolerant non-agricultural species, extends AMF research beyond traditional crops, offering insights into ecological restoration. These findings advance sustainable agriculture by demonstrating how targeted AMF inoculation can reduce reliance on chemical fertilizers while enhancing productivity and resilience across diverse plant systems.",
"discussion": "4 Discussion This study reveals the potential of F. mosseae as a plant growth-promoting fungus (PGPF) that profoundly impacts nutrient uptake and physiological performance in a range of plant species. The results indicated that fungal inoculation was incredibly beneficial for biomass accumulation, nutrient concentrations, and stress resilience, which validates the current avalanche of research on the effects of AMF. The increase in biomass yield across all tested plant species was most notable in rice and sesame because of increased nutrient acquisition mediated by F. mosseae . One of the primary mechanisms by which F. mosseae promotes plant growth is improving nutrient availability ( Ain et al., 2024 ). Specifically, the fungus enhances the ability of plants to access nutrients in the soil, particularly those that are less mobile, such as phosphorus and nitrogen ( Sanhueza et al., 2024 ). This might be due to its ability to extend the root zone by establishing extensive hyphal networks that effectively increase the fungus’s access to immobile nutrients, such as phosphorus and potassium ( Ain et al., 2024 ). The significant improvements in N and P uptake in Table 2 agree with those of previous studies ( Bisht and Garg, 2022 ; Hussain et al., 2021 ; Zhu et al., 2024 ), demonstrating that AMF improves the uptake of soil-bound nutrients and enhances nutrient use efficiency ( Table 2 ). Specifically, this interplay between fungal colonization and root growth promotion is significant in species with more extensive root systems, including rice and Mexican hat plants. Interestingly, the patterns observed in the uptake of nutrients in this study also help to explain how different plant species respond to fungal inoculation. In the case of the Mexican hat plant, a species known for its tolerance to abiotic stress, Mg (0.45 mg/kg) and Ca (1.13 mg/kg) uptake enormously increased compared to the control (0.34 mg/kg, 0.88 mg/kg), respectively. Concerning the reported improvements in nitrogen, potassium, calcium, and magnesium uptake, and the assertion that F. mosseae typically excels in phosphorus uptake only: While it is true that AM fungi are renowned for enhancing phosphorus uptake, recent literature has also shown that F. mosseae can influence the uptake of other nutrients ( Shi et al., 2021 ; Wu et al., 2024 ). In our experiments, the enhanced uptake of N, K, Ca, and Mg may be partly attributed to the fungus-induced improvements in root architecture and overall plant vigor, which in turn facilitate better soil exploration and nutrient acquisition. Our results implied that AMF inoculation facilitates plant growth by promoting the uptake of secondary macronutrients essential for plant physiological stability. This is consistent with studies on AMF-induced stress tolerance, in which the pivotal role of Mg in photosynthetic efficiency and chlorophyll synthesis has been demonstrated ( Hamzehzadeh et al., 2024 ; Wahab et al., 2023 ; Ye et al., 2019 ). While this study primarily focused on the effects of F. mosseae on nutrient uptake and root development, it is possible that the fungus also influences plant growth through the production of growth-regulating hormones such as auxins and cytokinins ( Yasmeen et al., 2024 ). In other studies involving mycorrhizal fungi, these hormones have been shown to enhance root development and overall plant vigor ( Zhang et al., 2019 ). Although hormonal regulation was not directly assessed in this study, the significant improvements in biomass and nutrient concentrations observed in the treatment groups suggest that hormonal interactions may also play a role in the growth-promoting effects of F. mosseae . Other measurements of chlorophyll fluorescence and photosynthetic performance also support the positive impact of F. mosseae on plant health. Previous research confirmed that AMF can alleviate oxidative stress and improve photosynthetic apparatus under suboptimal conditions ( Chauhan et al., 2022 ; Mo et al., 2016 ). However, differences in chlorophyll content and photosynthetic rates among species observed in this study demonstrate the complexity of AMF-plant interactions and underscore the necessity of species-specific application strategies. As shown in Figure 2 , the results of root development revealed that inoculated plants developed vigorous root systems with greater length and density. This improved root system development can be attributed to mycorrhizal colonization of the roots by F. mosseae ( Figure 4 ) ( Huang et al., 2019 ). The fungus facilitates nutrient exchange by enhancing the root surface area and improving the ability of plants to absorb water and nutrients. This process is a well-documented mechanism through which AMF promotes plant growth, particularly in nutrient-poor soils ( Chen et al., 2017 ). This is of great significance given the importance of roots for nutrient acquisition in nutrient-poor soils and for species that use extensive root networks, such as Egyptian peas, Mexican hat plants, and sorghum. F. mosseae has excellent potential as a biofertilizer in regions with degraded soils or low access to chemical fertilizers because the synergy of F. mosseae on root architecture and nutrient uptake efficiency has been reaffirmed. One of these promising findings also raises intriguing patterns that warrant further investigation. For example, although F. mosseae significantly increased most species’ growth and nutrient uptake, the magnitude of the increase differed. The source of this variability may be differences in the morphology of the root, compatibility with fungal symbionts, or inherent nutrient requirements of a particular organism ( Bever, 2015 ). Furthermore, slight differences in Mg uptake between species indicate that while fungal inoculation may differentially partition nutrients, the existing literature needs to explore this topic. The present study presents the ecological potential and potential for agronomic use of F. mosseae ; further work is needed to develop an appropriate application. Co-inoculation of AMF with other plant growth-promoting microorganisms (PGPMs) may synergistically affect soil respiration, nutrient cycling, and plant resistance. In addition, long-term field trials are necessary to evaluate the sustainability and scalability of integrating AMF in various cropping systems. Finally, the results of the investigations showed that F. mosseae has significant potential for stimulating the growth, nutrient uptake, and physiological performance of many plant species. Such findings indicate that it can be integrated into agricultural practices as an eco-friendly farming mode instead of chemical use. F. mosseae provides a pathway towards sustainable agricultural systems through improvements in crop productivity without loss of soil health. Nevertheless, it will not reveal the full potential of its interactions with plants, soils, and other microbial communities under changing environments."
} | 4,023 |
26347861 | PMC4542537 | pmc | 4,278 | {
"abstract": "The aromatic compounds cinnamic and p -hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli , Saccharomyces cerevisiae , Pseudomonas putida , and Streptomyces lividans . The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l -phenylalanine ( l -Phe) and l -tyrosine ( l -Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l -Phe or l -Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.",
"conclusion": "Conclusion and Outlook The development of microbial strains for the production of CA and pHCA from simple carbon sources involves extensive engineering of cellular metabolism. In addition to improving production characteristics, these modifications will likely also cause unexpected alterations to the cell’s physiology. System biology approaches offer the opportunity of better understanding the consequences of genetic modifications and the responses to various stress factors during the production stage. The application of omics-based approaches, such as transcriptomics, proteomics, fluxomics, and metabolomics, provides a comprehensive view of the cell’s physiology response to various genetic modifications as well as environment factors, such as product toxicity. For the case of microbial strains for the production of CA and pHCA, there is a lack of studies based on omics approaches. However, there are some reports focusing on the study of strains modified for the production of precursors of aromatic acids. In one report, proteome analysis was performed to understand the effect of inactivating a PYR kinase PykF in E. coli . Among proteins differentially expressed in the mutant strain, some were related to E4P synthesis and the common aromatic pathway, suggesting a higher capacity for aromatics synthesis (Prabhakar et al., 2007 ). Transcriptome analysis was performed to compare a PTS + and a PTS − glucose + \n E. coli strain modified for l -Phe production. Among differentially expressed genes, it was found that operon acs - actP that is involved in acetate consumption was upregulated in the PTS − glucose + strain (Báez-Viveros et al., 2007 ). This response is consistent with the lower level of acetate accumulation in culture medium observed for strain PTS − glucose + when compared to PTS + . These results provide useful data that helps in identifying genetic targets for strain improvement. Future studies focused on characterizing CA and pHCA production strains will likely identify novel targets for strain optimization. Aromatic acids CA and pHCA are valuable chemicals having direct applications and serving also as precursors for the synthesis of a large number of useful compounds. During the last years, various microbial hosts have been modified by metabolic engineering to generate production strains. These efforts have been fundamental for defining strain development strategies and for identifying factors that limit productivity. In contrast to other biotechnological products where a single microbial host is usually employed, for the case of CA and pHCA production, several different species show promise as production platforms. As reviewed here, E. coli , S. cerevisiae , P. putida , and S. lividans display particular characteristics that can favor aromatics acids production. Although much progress has been made with regard to production strain construction and process development, the yields of aromatic acids are still low when compared to other aromatic products (Bongaerts et al., 2001 ). An important factor limiting productivity is the toxicity of CA and pHCA. In this regard, studies identifying genes encoding efflux systems in E. coli and P. putida S12 enable a better understanding of the processes involved in mitigating aromatic acids toxicity (Kieboom et al., 1998 ; Van Dyk et al., 2004 ). The overexpression of these genes in each organism clearly increases resistance to toxic compounds. It remains to be determined if the solvent-tolerance trait can be transferred to a different species. The use of an omics approach to determine the transcriptional response to CA and pHCA should prove to be valuable for identifying systems that participate in toxic resistance in other microbial species. Generating a single product is usually the expected outcome in a biotechnological production system. In microbial strains engineered to produce aromatic acids from simple carbon sources, it has been shown that synthesis of CA as only product is possible, as a result of PAK specificity toward l -Phe. However, this is not the case for pHCA, since known TAL enzymes can also employ l -Phe as substrate. Therefore, pHCA is produced always with a certain amount of CA. Although downstream processing could be employed to separate pHCA from CA, this approach would result in increased production costs. Another solution to this issue could be based on applying protein engineering methods to modify substrate specificity of a TAL enzyme for reducing or abolishing CA production, while maintaining high-catalytic activity to increase production of pHCA. As an alternative, the search for novel TAL proteins in natural diversity has the potential for finding enzymes having substrate specificity only toward l -Tyr. Microbial strains having the capacity for producing CA or pHCA have been employed as platforms for the synthesis of various phenylpropanoid compounds. These include simple phenylpropanoids as well as lignoids, flavonoids, coumarins, and other related compounds (Figure 2 ) (Dixon and Steele, 1999 ). These plant metabolites have been shown to have pharmacological activities, such as antioxidants, anticancer, antiviral, anti-inflammatory, anti-nitric oxide production and antibacterial agents, among others (Dhanalakshmi et al., 2002 ). The microbial production of these compounds represents an attractive alternative to plant tissue extraction processes. However, at present, these microbial strains produce a low level of these plant compounds. It can be expected that some of the metabolic engineering strategies applied to CA and pHCA production strains, as reviewed here, should provide a basis for the future improvement of microbial strains that synthesize useful plant metabolites. Figure 2 Plant metabolites produced from CA and pHCA by engineered microbial strains .",
"introduction": "Introduction Bacteria and plants have the natural capacity for synthesizing a large number of aromatic compounds from simple carbon sources. The shikimate or common aromatic pathway is the main central metabolic branch leading to several biosynthetic pathways that produce various aromatic metabolites (Figure 1 ). The aromatic amino acids l -phenylalanine ( l -Phe), l -tyrosine ( l -Tyr), and l -tryptophan ( l -Trp) are primary metabolites synthesized from simple carbon sources by plants and bacteria. Secondary metabolites, such as phenylpropanoids, are derived from l -Phe and l -Tyr and are produced mainly by plants. The phenylpropanoid acids cinnamic acid (CA) and p -hydroxycinnamic acid (pHCA), also known as coumaric acid, are two metabolites having nutraceutical and pharmaceutical properties (Chemler and Koffas, 2008 ). They also have applications as precursors of chemical compounds and materials, such as high-performance thermoplastics (Kaneko et al., 2006 ; Sariaslani, 2007 ). Figure 1 Central metabolism, aromatics biosynthetic pathways, and transport pathways from engineered E. coli . Dashed arrows indicate multiple enzyme reactions. EI, PTS enzyme I; HPr, PTS phosphohistidine carrier protein; EIIA, PTS glucose-specific enzyme II; PTS IICB Glc , integral membrane glucose permease; GalP, galactose permease; XylFGH, xylose transport proteins, AraFGH, arabinose transport proteins; DAHPS, DAHP synthase; aroG fbr , gene encoding a feedback-inhibition-resistant version of DAHPS; tktA , transketolase; tyrB , tyrosine aminotransferase gene; PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; C4H, cinnamate 4-hydroxylase; AaeXAB, efflux pump from E. coli ; SprABC, efflux pump from P. putida ; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; R5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; X5P, xylulose-5-phosphate; PYR, pyruvate; AcCoA, acetyl-CoA; TCA, tricarboxylic acids. Both CA and pHCA are present in plant tissues at a low concentration. Therefore, complex procedures must be employed for their extraction and the yields are usually low. For these reasons, alternative production schemes are being explored. Several microbial species currently employed in biotechnological processes, possess part of the pathways required for CA and pHCA synthesis from simple carbon sources. Thus, by applying genetic engineering techniques to various microbial species, it has been possible to develop production strains with the novel capacity for synthesizing phenylpropanoid acids (Nijkamp et al., 2007 ; Vannelli et al., 2007a ; Limem et al., 2008 ). In this review, we focus on recent studies related to the application of genetic engineering strategies for the development of strains derived from Escherichia coli , Pseudomonas putida , Streptomyces lividans , and Saccharomyces cerevisiae for the production of the phenylpropanoid acids CA and pHCA. Production process development issues, such as product toxicity and carbon source utilization, are also discussed."
} | 2,734 |
30250477 | PMC6139337 | pmc | 4,279 | {
"abstract": "Arbuscular mycorrhizal (AM) fungi have become an attractive target as biostimulants in agriculture due to their known contributions to plant nutrient uptake and abiotic stress tolerance. However, inoculation with AM fungi can result in depressed, unchanged, or stimulated plant growth, which limits security of application in crop production systems. Crop production comprises high diversity and variability in atmospheric conditions, substrates, plant species, and more. In this review, we emphasize that we need integrative approaches for studying mycorrhizal symbioses in order to increase the predictability of growth outcomes and security of implementation of AM fungi into crop production. We briefly review known mechanisms of AM on nutrient uptake and drought tolerance of plants, on soil structure and soil hydraulic properties. We carve out that an important factor for both nutrient availability and drought tolerance is yet not well understood; the AM effects on soil hydraulic properties. We gave special emphasis to circular references between atmospheric conditions, soil hydraulic properties and plant nutrient and water uptake. We stress that interdisciplinary approaches are needed that account for a variability of atmospheric conditions and, how this would match to mycorrhizal functions and demands in a way that increased plant nutrient and water uptake can be effectively used for physiological processes and ultimately growth. Only with integrated analyses under a wide range of growing conditions, we will be able to make profound decisions whether or not to use AM in particular crop production systems or can adjust culture conditions in ways that AM plants thrive."
} | 423 |
36274956 | PMC9579507 | pmc | 4,280 | {
"abstract": "Summary Root exudates and rhizosphere microorganisms play key roles in the colonization of toxic plants under climate change and land degradation. However, how root exudates affect the rhizosphere microorganisms and soil nutrients of toxic plants in degraded grasslands remains unknown. We compared the interaction of soil microbial communities, root exudates, microbial carbon metabolism, and environmental factors in the rhizosphere of toxic and non-toxic plants. Deterministic processes had a greater effect on toxic than non-toxic plants, as root exudates affected rhizosphere microorganisms directly. The 328 up-regulated compounds in root exudates of toxic plants affected the diversity of rhizosphere microorganisms. Rhizosphere bacteria-enriched enzymes were involved in the phenylpropanoid biosynthesis pathway. Root exudates of toxic plants form complex networks of rhizosphere microorganisms, provide high rhizosphere nutrients, and increase microbial carbon metabolism. The interaction between root exudates and rhizosphere microorganisms is the key mechanism that enables toxic plants to spread in degraded grassland habitats.",
"conclusion": "Conclusions Overall, the convergence of root functions in toxic plants could improve their adaptability in different degraded grasslands. Toxic plants secrete more bio-active compounds beneficial to plant growth than non-toxic plants. These compounds include naphthoquinones, terpenoids, phthalates, amino acids, coumarin, and flavones, which alter microbial diversity. The assembly of rhizosphere microorganisms was mainly a stochastic process. The deterministic process (niche theory) also contributed and had a greater effect on toxic than non-toxic plants, as root exudates directly affected the assembly of rhizosphere microorganisms. Compared with bulk soil, the rhizosphere had higher nutrients and greater microbial C metabolism activity. In addition, the network of the microbial community in the rhizosphere of toxic plants was more complex and had higher positive correlations among microorganisms than bulk soil and rhizosphere of non-toxic plants, indicating greater interaction and niche sharing potential among microorganisms in the rhizosphere of toxic plants. Bacteria in the rhizosphere of toxic plants were enriched in the phenylpropanoid biosynthesis pathway. It is concluded that the strong interaction between root exudates and microorganisms is the key mechanism for the divergent habitat adaptation of toxic plants in different degraded grasslands, supporting above-ground plant growth and reproduction.",
"introduction": "Introduction The expansion of plants that are toxic to grazing animals has become a global concern ( Colautti and Barrett, 2013 ; Savary et al., 2019 ), as it is causing a substantial loss in grassland ecosystem services under climate change and anthropogenic activities (Humphries et al., 2021). Identifying the driving forces that enable the infestation of toxic plants would be beneficial in grassland management. Recent research on the spread of toxic plants has focused mainly on factors such as grazing livestock and climate change ( Ricciardi et al., 2017 ; Liu et al., 2020 ). However, how toxic plants adapt to the poor soil conditions of degraded grassland is unknown. The self-reinforcement ability of toxic plants is closely associated with the root system in degraded land ( Wagg et al., 2011 ; Liu et al., 2018 ), as root exudates play a key role in plant growth and in the structure, diversity, and functioning of the rhizosphere microbial community ( Koprivova et al., 2019 ; Zhou et al., 2021 ; Gross, 2022 ). For example, the tryptophan-derived molecule, camalexin, inhibited fungal pathogens and specific bacteria in Arabidopsis , thereby, altering the root microbiota ( Koprivova et al., 2019 ). Umbelliferone in Stellera chamaejasme influenced cell division and induced membrane lipid peroxidation, which inhibited the growth of plants ( Yan et al., 2016 ). Toxic plants produce a broad range of secondary metabolites ( Shang et al., 2012 ; Yan et al., 2016 ), which have a substantial impact on the root environment ( Goldschmidt et al., 2018 ). Root exudates are the main driving forces regulating the diversity and metabolic activities of rhizosphere microorganisms during plant growth ( De Vries et al., 2019 ). It was reported that wild oat ( Avena fatua ) had a more complex network in the rhizosphere than bulk soil, and microbial diversity decreased as network size decreased ( Shi et al., 2016 ). Rhizosphere with a highly selective environment could promote species taxonomic co-occurrence, which could be indicative of increased metabolic specifics ( Fan et al., 2018 ; Wang et al., 2020 ). It has been reported that microbial communities in the rhizosphere are shaped by deterministic processes (niche) and in bulk soil by stochastic processes (neutral) ( Dumbrell et al., 2010 ; Mendes et al., 2014 ; Lima-Mendez et al., 2015 ). As habitat heterogeneity declines at smaller scales, a more apparent contribution of stochastic over deterministic processes is evident ( Legendre et al., 2009 ). Toxic plants are capable of spreading and dominating degraded grasslands with nutrient-poor soil, unlike non-toxic plants ( Figure S1 ) ( Sui et al., 2015 ; Yao et al., 2019 ; Humphries et al., 2021 ). We hypothesized that the interaction between rhizosphere microorganisms and root exudates facilitates the spread of toxic plants in degraded grasslands. To test this hypothesis, we measured microbial carbon metabolic activity, compounds, and functions of root exudates, and integrated taxonomic and functional data to describe the soil microbial communities of toxic and non-toxic plants in degraded alpine grasslands. The study was guided by the following predictions: (1) specific root exudates mediate the composition, network and carbon metabolic activity of rhizosphere microorganisms of toxic plants; (2) deterministic processes could explain the assembly of microorganisms in the rhizosphere of toxic plants better than stochastic processes; (3) the network complexity of the microbial community in toxic plants is greater than in non-toxic plants; and (4) the interaction of root exudates and rhizosphere microorganisms is the key factor that enables the toxic plant to spread in degraded grasslands.",
"discussion": "Discussion Assembly of rhizosphere bacterial community The microbial community assemblies are shaped by a multitude of trophic influences, which depend upon biological diversity ( Caruso et al., 2011 ; Lebeis et al., 2015 ). Neutral theory predicts that if limited dispersal and demographic stochasticity are the main drivers of community dynamics, then the random pattern in species and spatial auto-correlation of the environment should be the main factors determining community structure ( Sloan et al., 2006 ; Mendes et al., 2014 ). In contrast, deterministic processes influence biodiversity and species composition, if niche partitioning interacts with environmental factors ( Chase, 2007 ). The present study indicated that the microbial assemblies of rhizospheres were shaped mainly by niche filtering, which supported our hypothesis. The selection at the functional level in the rhizosphere was based on deterministic processes according to the niche-based theory. Furthermore, habitat and species affected the composition of microbial community structure. This trend was evident for toxic plants, which indicated that the rhizosphere of toxic plants had a strong influence in shaping the bacterial community in degraded grassland. Three densities of L. virgaurea were identified in the present study. There was a high death rate with high density, which may have been caused by autotoxicity and/or ‘self-thinning’. Autotoxicity is allelopathy in which an individual inhibits the growth of other individuals of the same species by releasing autotoxins ( Singh et al., 1999 ). Some autotoxins, including phenolics, omilactone B, artemisinin, phenolic acids, and cyclic hydroxamic acids ( Ni et al., 2012 ), inhibit or delay the germination and growth of conspecific plants ( Miller, 1996 ). Owing to overlapping and interference mechanisms, roots provide a specific micro-habitat for the proliferation of specific soil microorganisms, with new interactions developing among colonizing microbes in densely colonized rhizospheres ( Petermann and Buzhdygan, 2021 ; Tian et al., 2022 ). Microbial network complexity in rhizosphere microorganisms of toxic plants Rhizosphere assemblages formed larger and more complex networks than bulk soil assemblages, which supported our prediction. The network modules likely resulted from microbe interactions or covariation in response to shared niches in the rhizosphere ( Shi et al., 2016 ; Wang et al., 2021 ), owing to better nutrition and greater microbial C metabolism activity than in bulk soil microbes. In contrast to rhizosphere microbes, networks in bulk soil remained relatively simple, which indicated that interactions or niche sharing were minimal. The low activity of soil bacteria was another reason for the lack of networks in bulk soil ( Fierer and Lennon, 2011 ). Within rhizosphere modules, 15 modules were identified, which further indicated that the rhizosphere microbial networks were more complex than in bulk soil. The related properties of network modules were closely related to soil multi-functionality, suggesting that microorganisms influenced soil nutrient cycling processes ( Wagg et al., 2019 ; Marqués-Gálvez et al., 2021 ). In the process of grassland degradation, soil nitrate nitrogen content increased, while ammonia nitrogen content decreased, which may be owing to enhanced nitrification and decreased denitrification by microorganisms. Studies have reported that low denitrification in soil may be related to plant interactions ( Dassonville et al., 2011 ). The root exudates of toxic plants contained higher contents of biologically active compounds (coumarin, cinnamic acid, flavones) than in non-toxic plants, and they were correlated positively with the dominant taxa in the module. Flavonoid compounds could interfere with bacterial respiration, especially with denitrification ( Bardon et al., 2014 ). Therefore, root exudates promoted the development of the niche occupied by dominant taxa, and greater interactions owing to the shared ecological niche, which resulted in more complex co-occurrence patterns in toxic than in non-toxic plants. This could explain the greater abundance of microorganisms in toxic than in non-toxic plants. In degraded sown grassland in this study, high-density L. virgaurea had a related exudate module, and the middle and low densities L. virgaurea had related bacterial modules ( Figure S10 ). It is probable that the root-associated microbial populations were determined not only by the available C sources but also by selective and inhibitory interactions ( Trivedi et al., 2020 ). Compared with high-density L. virgaurea , the rhizosphere microbes of low-density L. virgaurea had less nutrients available. The roots of low-density L. virgaurea provided less C for their rhizosphere microorganisms, and, as a result, the rhizosphere microorganisms may have been affected by nutrient stress. Therefore, the stronger the positive interaction between microbes, the more restricted the dispersal of species ( Table S1 ), which was consistent with the stress gradient hypothesis ( David et al., 2020 ), and further emphasized the importance of root exudates to the assemblies of rhizosphere microorganisms. Root exudates affect rhizosphere microorganisms In the present study, root exudates of toxic plants were correlated positively with rhizosphere microorganisms ( Figure S11 ), which supported the first and fourth hypotheses. This relationship has been observed in many plant species and soil types ( Liu et al., 2020 ); however, this is not always the case. Roots of Morina kokonorica and Aconitum pendulum released unique compounds that were not correlated with rhizosphere microorganisms. This may have been caused by sampling time or plant communities, as the adjacent plants or different growth stages of plants release complex root exudates that enrich different rhizosphere microorganisms ( Kong et al., 2018 ). When roots of Lupinus albus mature, organic acids are released that decrease the pH of the soil to inhibit bacteria ( Weisskopf et al., 2006 ). In the current study, the pH and organic matter and nitrogen contents in the rhizosphere were higher than in bulk soil. In addition, the SEM demonstrated that pH influenced the bacterial communities directly. As reported by Hermans et al. (2020) , an increase in pH affected the solubility of elements, promoted the growth of plants, and led to an increase in root exudates and soil organic matter content. Furthermore, soil surface nutrient cycling was enhanced, as plants extracted more nutrients (K and NO 3 ), which were decomposed and mineralized at the surface ( Liddicoat et al., 2019 ). The concentrations of coumarin derivatives, naphthoquinones, salicylic acid, cinnamic acid, and terpenoids were greater in the root exudate of toxic than non-toxic plants in the present study. This is consistent with the report that toxic plants have improved chemical defense substances (terpenoids and phenols), potentially contributing to their reproductive success. Mendes et al. (2011) reported that Proteobacteria and Actinobacteria were associated with disease suppression, and Proterobacteria was correlated positively with bio-active compounds of root exudates in toxic plants ( Figure S8 ). In addition, the relative abundances of Bradyrhizobium , Pseudomonas , Rhizobium , and Sphingomonas , mainly plant growth promoting rhizobacteria (PGPR), were greater in toxic than non-toxic plants. The significantly up-regulated compounds in this study were correlated positively with bacterial phyla ( Figure S8 ). Root exudates have been shown to affect the composition of the rhizosphere microbial community. Salicylic acid plays an important role in plant resistance to pathogens ( Leibold and McPeek, 2006 ), and coumarin is beneficial to the interaction among probiotics ( Voges et al., 2019 ; Harbort et al., 2020 ). Huang et al. (2019) reported that Arabidopsis produced specialized triterpenes that shaped and customized the microorganisms within and around its roots and maintained specific microbiota. Naphthoquinones possess antimicrobial activity ( Brigham et al., 1999 ), are derived from phenylpropanoid and isoprenoid precursors ( Gaisser and Heide, 1996 ), and enhance the self-growth and defense of the next-generation of plants ( Hu et al., 2018 ). These compounds may play a similar role in toxic plants by inhibiting the growth of nearby plants, enriching beneficial bacteria, and helping plants resist pathogens. The effects of bio-active compounds in root exudates of toxic plants on rhizosphere microorganisms warrant further studies. Life cycle influenced the adaptation of toxic plants According to our survey, L. virgaurea has a long pre-flowering vegetative growth stage, generally 3–6 years, and produces rhizomes, which store nutrients for clonal growth. In the current study, L. virgaurea had a wider niche than other toxic plants; perhaps the spatial colonization mechanisms of clonal plants are driven by the growth of the rhizome. The rhizosphere bacterial communities had higher positive correlations with root exudates in L. virgaurea and Pedicularis kansuensis than with other toxic plants. The samples were taken in August, which was the peak time for the development of plants and L. virgaurea , a perennial, was at the stage of asexual reproduction. In addition, P. kansuensis , an annual, parasitizes the host during growth and reproduction ( Sui et al., 2015 ). As root exudates could be affected by the developmental stage of plants ( Zhalnina et al., 2018 ), further studies are needed to determine the effect of the growth stage on microbial communities. Conclusions Overall, the convergence of root functions in toxic plants could improve their adaptability in different degraded grasslands. Toxic plants secrete more bio-active compounds beneficial to plant growth than non-toxic plants. These compounds include naphthoquinones, terpenoids, phthalates, amino acids, coumarin, and flavones, which alter microbial diversity. The assembly of rhizosphere microorganisms was mainly a stochastic process. The deterministic process (niche theory) also contributed and had a greater effect on toxic than non-toxic plants, as root exudates directly affected the assembly of rhizosphere microorganisms. Compared with bulk soil, the rhizosphere had higher nutrients and greater microbial C metabolism activity. In addition, the network of the microbial community in the rhizosphere of toxic plants was more complex and had higher positive correlations among microorganisms than bulk soil and rhizosphere of non-toxic plants, indicating greater interaction and niche sharing potential among microorganisms in the rhizosphere of toxic plants. Bacteria in the rhizosphere of toxic plants were enriched in the phenylpropanoid biosynthesis pathway. It is concluded that the strong interaction between root exudates and microorganisms is the key mechanism for the divergent habitat adaptation of toxic plants in different degraded grasslands, supporting above-ground plant growth and reproduction. Limitations of the study Climate warming may affect the metabolism of plants, increase the defense substances and promote the invasion of weeds ( Rice et al., 2021 ). In the present study, increased content of defensive substances was present in the roots of toxic plants in a degraded alpine grassland on the Qinghai-Tibetan Plateau. These substances have a direct effect on the assembly process of rhizosphere microorganisms, which may have a positive effect on the reproduction of toxic plants. Studies that support this argument directly are few. In addition, since toxic weeds are usually perennial plants, it is not clear whether responses are similar at different stages of growth. Further studies on different growth stages of toxic weeds are needed, in particular in the later stages. Whether rhizosphere microorganisms and root exudates are similar in different growth stages also requires further research."
} | 4,596 |
36838385 | PMC9959488 | pmc | 4,282 | {
"abstract": "Analyzing microbial communities using metagenomes is a powerful approach to understand compositional structures and functional connections in anaerobic digestion (AD) microbiomes. Whereas short-read sequencing approaches based on the Illumina platform result in highly fragmented metagenomes, long-read sequencing leads to more contiguous assemblies. To evaluate the performance of a hybrid approach of these two sequencing approaches we compared the metagenome-assembled genomes (MAGs) resulting from five AD microbiome samples. The samples were taken from reactors fed with short-chain fatty acids at different feeding regimes (continuous and discontinuous) and organic loading rates (OLR). Methanothrix showed a high relative abundance at all feeding regimes but was strongly reduced in abundance at higher OLR, when Methanosarcina took over. The bacterial community composition differed strongly between reactors of different feeding regimes and OLRs. However, the functional potential was similar regardless of feeding regime and OLR. The hybrid sequencing approach using Nanopore long-reads and Illumina MiSeq reads improved assembly statistics, including an increase of the N50 value (on average from 32 to 1740 kbp) and an increased length of the longest contig (on average from 94 to 1898 kbp). The hybrid approach also led to a higher share of high-quality MAGs and generated five potentially circular genomes while none were generated using MiSeq-based contigs only. Finally, 27 hybrid MAGs were reconstructed of which 18 represent potentially new species—15 of them bacterial species. During pathway analysis, selected MAGs revealed similar gene patterns of butyrate degradation and might represent new butyrate-degrading bacteria. The demonstrated advantages of adding long reads to metagenomic analyses make the hybrid approach the preferable option when dealing with complex microbiomes.",
"introduction": "1. Introduction Biogas production by anaerobic digestion (AD) provides a sustainable option to transform organic waste into a renewable energy carrier. Under anoxic conditions, organic matter is converted to short-chain fatty acids (SCFAs) as intermediates, and finally to methane and carbon dioxide, catalyzed by different microbes in four steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. The first three steps are facilitated by bacteria, whereas the last step is performed by archaea. There are multiple types of biogas reactors. The continuous stirred tank reactor (CSTR) is the most common type for agricultural biogas plants in Germany. The organic loading rate (OLR) is a key parameter in continuous AD processes. At high OLRs, large substrate amounts are fed to an AD reactor, and consequently high methane productivity can be expected. However, high OLRs can also lead to process instabilities and even process failure [ 1 ]. The stability of the AD process depends on the interaction of microbes of different functional groups [ 2 ]. An important prerequisite to control the process stability is to predict compositional and functional changes of the microbial community as a function of operational parameters. Community composition and complexity as well as operational parameters influence community dynamics [ 2 , 3 ]. For instance, the feeding regime can affect the process stability as well as the microbial community function [ 1 , 4 , 5 , 6 ]. The continuous feeding mode is commonly applied in full-scale digesters to obtain stable biogas production [ 7 ]. However, several studies showed positive effects of discontinuous feeding (e.g., one feeding per day) compared to continuous feeding [ 1 , 8 , 9 , 10 ]. Bonk et al. observed that discontinuous feeding leads to higher relative abundances of Methanosarcina [ 1 ]. This methanogen is more adapted to higher acetic acid concentrations and more resistant to low pH values compared to the common acetoclastic methanogen Methanothrix [ 5 ]. A higher share of Methanosarcina increased the resilience against organic overloading [ 1 ]. Some products of the acidogenesis and acetogenesis steps, such as acetate, formate, H 2 , and CO 2 as well as methyl compounds, can be utilized by methanogenic archaea directly. In contrast, propionate and butyrate are acidogenesis products that first need to be converted into acetate, CO 2 , and H 2 or formate [ 11 ]. In the whole AD process, syntrophic butyrate and propionate oxidation during acetogenesis is a bottleneck because of the low abundances of butyrate- and propionate-oxidizing bacteria and their sensitivity to environmental changes [ 2 , 12 , 13 ]. Disturbances of the AD process can lead to the accumulation of SCFA and consequently process instability [ 12 , 14 , 15 , 16 ]. Thus, the cooperation between bacteria and methanogenic archaea is essential for stable process performance. Typical propionate- and butyrate-oxidizing bacteria are Syntrophobacter , Pelotomaculum, Smithella , and Syntrophomonas [ 12 , 17 ]. In AD processes, hydrogenotrophic methanogens depend on hydrogen-producing bacteria, and inversely, syntrophic bacteria can only grow when the hydrogen partial pressure is kept low by hydrogenotrophs. For instance, fatty acid-degrading bacteria that use the β-oxidation pathway, such as Syntrophomonadaceae , grow in syntrophy with hydrogen-consuming microbes [ 6 , 18 ]. Besides acetoclastic methanogenesis catalyzed by Methanothrix or Methanosarcina , syntrophic acetate oxidation (SAO) is an alternative acetate sink in AD. Under mesophilic conditions, Thermacetogenium phaeum , Pseudothermotoga lettingae , Tepidanaerobacter acetatoxidans , Clostridium ultunense , and Syntrophaceticus schinkii [ 19 ] are key players of SAO whereby acetate is converted into H 2 and CO 2 [ 20 , 21 ]. In SAO, both the methyl and carboxyl groups of acetate are oxidized to CO 2 , and H 2 is formed, but the reaction is unfavorable in the absence of hydrogenotrophic methanogens. Therefore, SAO is also an example of the interdependencies between bacteria and archaea in AD [ 22 ]. Two metabolic pathways for anaerobic propionate degradation have been described: the methylmalonyl-CoA pathway and the C6-dismutation pathway. Examples of syntrophic propionate-oxidizing bacteria (SPOB) that use the methylmalonyl-CoA pathway are Syntrophobacter and Pelotomaculum [ 23 ]. These SPOB degrade propionate directly to acetate and CO 2 . Propionate is first carboxylated to methylmalonyl-CoA, which is further converted via succinate, fumarate, and malate to oxaloacetate, followed by two decarboxylation steps to pyruvate and eventually to acetyl-CoA [ 24 , 25 ]. In contrast, Smithella uses the C6-dismutation pathway converting propionate to acetate and n -butyrate as an intermediate [ 26 ]. Butyrate is then further degraded to acetate via the β-oxidation pathway [ 27 ]. For the last AD step, methanogenesis, three important pathways have been described: the acetoclastic, the hydrogenotrophic, and the methylotrophic pathway. In the acetoclastic pathway, acetate is activated to acetyl-CoA and then cleaved into CO 2 and a methyl group, which is further reduced to CH 4 [ 28 , 29 ]. Methanothrix and Methanosarcina are the only two genera performing acetoclastic methanogenesis. Most of the other known methanogens utilize the hydrogenotrophic pathway, which accounts for the remaining portion of methane production resulting from the reduction of carbon dioxide with hydrogen/formate [ 30 , 31 ]. In this study, Illumina-based shotgun metagenome sequencing was used to characterize and compare compositional and overall functional profiles of reactor microbiomes being subjected to different feeding regimes and OLRs. By restricting the substrate to SCFA, we aimed to select microbiomes that are less diverse than common AD microbiomes and facilitate the functional analysis of acetogenesis and methanogenesis as the last two AD steps. To analyze the genetic profile of individual members of the microbial community (particularly the bacterial community), Illumina-based short-read and Oxford Nanopore-based long-read assemblies were compared with respect to the recovery of high-quality metagenome-assembled genomes (MAGs). Read length is a limitation of the Illumina platform but the high quality is an advantage. Oxford Nanopore reads are long, but the quality is much lower with an error rate of up to 15%, compared to the Illumina error rate of 0.473% (median) [ 32 , 33 , 34 , 35 ]. By applying a hybrid assembly approach, the disadvantages of both techniques can be compensated [ 36 , 37 , 38 ], hence we expected that the completeness of the MAGs increases compared to Illumina-based MAGs. Additionally, the identification and analysis of MAGs can help predict important functional groups [ 39 , 40 , 41 ].",
"discussion": "4. Discussion By using metagenome approaches, we analyzed composition and functional potential of microbial communities in anaerobic digesters under two different feeding regimes at high and low OLRs, with a focus on the conversion of SCFAs to methane. We focused the analysis on selected pathways of particular interest (SCFA oxidation and methanogenesis) and detected associated genes in reads and MAGs. MAGs were reconstructed and annotated to shed light on uncharacterized taxa. The goal was to characterize the full community and identify the putative functional role of not yet described species. Previous analysis of the methanogenic reactor communities by mcrA -gene based terminal restriction fragment length polymorphism (T-RFLP) analysis had identified Methanothrix and Methanomicrobiaceae as the main constituents, and an increased share of Methanosarcina under discontinuous feeding [ 1 ]. These findings were confirmed with metagenome-based analysis in which Methanothrix and Methanoculleus were the main archaeal genera, and the share of Methanosarcina was increased under discontinuous feeding (R-2, R-3, R-5, Figure 1 c), a shift that was more pronounced under high OLRs (R-5, [ 1 ]), where Methansarcina partially replaced Methanothrix . It is known that Methanothrix is more sensitive to changing environmental conditions and stress conditions (feeding regime or OLR) than Methanosarcina and Methanoculleus [ 5 , 77 , 78 , 79 , 80 ]. Hence, stress tolerance of AD systems can be promoted if Methanosarcina can be enriched in the reactor at the expense of the more sensitive species, increasing the functional resilience of the methane production process [ 1 ]. Discontinuous feeding was shown to increase the relative abundance of Methanosarcina , and Bonk et al. [ 1 ] hypothesized that the dynamic environment established by discontinuous feeding provides temporal niches that enable the observed shift in the archaeal community. The bacterial communities were unique for all reactors, even for reactors R-2 and R-3, which were biological replicates, and no clear trends could be observed regarding OLR and feeding regime ( Figure 1 d). This has been reported before, including similar values for diversity and evenness of the bacterial communities based on 16S rRNA gene amplicon sequencing data [ 1 ]. Using metagenomic data to assess community composition circumvents any PCR biases and, by targeting bacteria and archaea simultaneously, enables the assessment of the ratio between both domains. The observed dominance of archaea was expected as the direct supply of SCFAs as substrate led to the establishment of only the last two steps of AD (i.e., syntrophic SCFA oxidation and methanogenesis), excluding the hydrolysis and acidogenesis steps driven by bacteria. Comparable ratios of bacteria to archaea were also found in other anaerobic digesters supplied with SCFAs [ 81 ]. Propionate and butyrate are degraded by bacteria in syntrophy with hydrogenotrophic methanogens, while acetate can either be directly utilized by acetoclastic methanogens or by acetate-oxidizing bacteria, again requiring a hydrogenotrophic methanogen as a syntrophic partner. Despite the observed variability in bacterial community composition, the overall reactor productivity was not affected, indicating the presence of functional redundancy in the bacterial communities. In low OLR reactors, the bacterial community was dominated by one or two bacterial genera, while high OLR reactors showed a more even distribution of the different genera, indicating that competition played a bigger role under nutrient-limiting conditions. An adaptation of the bacterial community due to an increase in OLR was also observed by Maus et al. [ 82 ]. Although being dominated by archaeal genera, the reactors in this study harboured diverse bacterial genera, many with relative abundances below 1%. Given that SCFAs were provided as the sole carbon sources, one could expect that most of these genera are involved in syntrophic SCFA oxidation. Syntrophobacter and Pelotomaculum were identified as known syntrophic oxidizers for propionate. Synthrophomonas was the only butyrate-oxidizing genus detected. Genera such as Cloacimonetes [ 83 , 84 , 85 ], Cryptanaerobacter [ 86 ], and Desulfovibrio [ 87 ] have been associated with the oxidation of propionic or butyric acid. Candidatus Cloacamonas acidaminovorans, for example, contains all the genes for propionic acid oxidation via methylmalonyl-CoA, but has not yet been isolated [ 83 ]. Besides these known and putative SCFA-oxidizers, our MAGs analysis uncovered many bacteria that were apparently not involved in SCFA oxidation. Similar observations have been made in AD reactors being fed with a single SCFA (acetate or propionate) [ 88 ]. Yeast extract was present in the medium in the latter study, potentially supporting the growth of other bacteria than SCFA oxidizers. However, this supplement was absent in our study, and hence cannot explain the observed bacterial diversity. Bacteria not directly utilizing the primary carbon source could either be involved in biomass turnover as scavengers or thrive on proteins, amino acids, polysaccharides, and lipids produced by primary consumers and associated syntrophic partners. Autotrophic methanogens can actively excrete amino acids into the medium [ 75 , 76 ], and amino acid exchange has been postulated in a synthetic coculture [ 89 ]. Furthermore, metabolite cross-feeding, especially between phylogenetically distant species, might be an important factor driving microbial community dynamics [ 90 ], and explain the observed diversity of bacterial taxa, including proteolytic fermenters and amino acid oxidizers not involved in SCFA oxidation. The primary consumers together with their syntrophic partners may hence be the source of excreted nutrients, which sustain a low abundance shadow microbial community of taxa not involved in the primary process. Overall, the microbial community composition at the genus level was more similar for reactors only differing in feeding regime, whereas those with different OLR harboured more distinct communities, indicating the importance of substrate quantity for shaping community composition. For example, the ratio of bacteria to archaea was mostly determined by the OLR but not by the feeding regime. The functional potential was similar between reactors despite different community composition. Hence, functional redundancy in SCFA degradation by known or unknown bacterial species could play an important role. By applying a higher OLR (reactors R-4 and R-5), the relative abundance of bacteria increased compared to the low OLR reactors R-1 to R-3. However, the genus Syntrophomonas , comprising typical butyrate-oxidizing bacteria [ 20 , 91 ], showed the highest abundance in the low OLR reactor R-1. Syntrophomonas was also described as the major syntrophic butyrate-oxidizing bacterium in a butyrate-fed system [ 92 ]. Its lower abundance in the other reactors indicates that other species took over its function there. Other species only appeared under high OLR conditions or increased their abundance. For instance, Corynebacterium was not found in R-2 and R-3 but had an abundance in the bacterial community of around 13% in R-5, and Mesotoga was present with a relative abundance of less than 1% in R-2 and R-3 but reached around 3% in R-5. Genera like unclassified Bacteroidetes , Candidatus Cloacimonas, Mesotoga, Desulfovibrio , or Endomicrobium were detected with higher abundance, but none of these taxa covered the complete butyrate degradation pathway. Pathway analysis showed that Desulfosporosinus , Clostridium , or Bacillus showed the same pathway coverage of selected genes as Syntrophomonas . However, the relative abundance of these genera was below 1% of the whole community. Thus, potential functional redundancy between yet undescribed taxa and known taxa might be present in our reactors. Candidatus Cloacimonas was detected mainly in the R-4 community with around 5%. Being frequently detected in AD processes [ 83 , 93 ], the reconstruction of a high quality MAG classified as Candidatus Cloacimonas and with 100% completeness (UFZ-4H1) provides the opportunity to identify its role in the process. However, regarding the main pathways analyzed in this study, there was no clear indication for its participation in syntrophic SCFA oxidation. For example, only three genes out of ten for propionate oxidation via the methonylmalonyl-CoA pathway were found ( Figure 3 ), although the complete pathway was reported for the genome of Candidatus C. aminoacidivorans [ 83 ]. Regarding typical propionate-degrading bacteria like Desulfotomaculum , Pelotomaculum , Smithella , and Syntrophobacter [ 20 ], only Syntrophobacter was detected with more than 5% in the bacterial community and only around 1.5% in the whole community of R-4 and R-5. The pathway analysis of selected genes of propionate oxidation showed a low coverage of these genes by Syntrophobacter . In contrast, Clostridium and Desulfotomaculum displayed a higher share of the typical propionate oxidation genes. A closer examination of Desulfotomaculum suggested that this genus is metabolically versatile, because it encodes many selected genes for acetate, butyrate and propionate oxidation. A single species of Desulfotomaculum has been described as syntrophic propionate oxidizer yet [ 94 ]. The results of Illumina reads as well as the analysis of MAGs showed that Methanothrix (UFZ-4H12) encoded all selected genes of the hydrogenotrophic methanogenesis pathway, besides the acetoclastic methanogenesis pathway. This finding was described already by Smith & Ingram-Smith [ 95 ]. As hydrogenotrophic methanogenesis activity of Methanothrix has not been described, it has been speculated that this genus can produce methane based on direct interspecies electron transfer [ 96 ]. However, as acetic acid was available in our reactors, Methanothrix most likely utilized the acetoclastic methanogenesis pathway. Read length is a limitation of the Illumina platform but the high quality is an advantage. Oxford Nanopore reads are long but the quality with an error rate up to 15% is much higher compared to the Illumina read quality [ 32 , 33 , 34 ]. By applying a hybrid assembly approach, the disadvantages of both techniques can be compensated. Even though Nanopore reads get more accurate due to improvements in technology, Illumina, for polishing of especially low-coverage MAGs, is advantageous [ 97 ]. We were expecting that the completeness of the genomes would increase compared to Illumina short read-based MAGs. Furthermore, as expected and as described in the literature e.g., [ 38 , 97 ] the contiguity of the MAGs was considerably increased by incorporating long reads. By the reconstruction of MAGs, several potentially new species could be identified. Focusing on high quality MAGs, a Syntrophobacteraceae species potentially involved in syntrophic SCFA oxidation (UFZ-4H3), three Syntrophomonadaceae species potentially involved in butyrate oxidation (UFZ-4H4, UFZ-1H11, and UFZ-1H7), and one Methanoculleus species potentially involved in hydrogenotrophic methanogenesis (UFZ-1H3) were identified ( Figure 3 ). The metabolic pathway analysis focused on the two key processes in the reactor: syntrophic SCFA oxidation as the utilization step of the provided carbon sources and methanogenesis as the syntrophically coupled process, providing the required electron sink. However, some of the high-quality MAGs could not be clearly assigned to these key processes (UFZ-4H14, UFZ-1H10, UFZ-4H10, UFZ-4H1, and UFZ-4H6; Figure 3 ). This indicates that the core community catalyzing the key processes is able to support a wider food web, explaining the observed high diversity. The discovered MAGs provide a first glimpse into this food web, potentially comprising metabolite cross-feeding and scavengers involved in biomass turnover. The high relative abundance of the archaeal populations limited the read numbers for bacterial populations, limiting the opportunity to reconstruct complete bacterial genomes of rare species. A higher number of bacterial reads would allow to reconstruct more complete genomes of potential new functional groups or species. Nevertheless, 13 medium- and high-quality MAGs could be obtained. The GTDBbk database classification leads to the assumption that some bacterial MAGs belong to the families Syntrophobacteraceae (UFZ-4H3, UFZ-4H4) and Cloacimonadaceae (UFZ-4H1) or the order Bacteroidales (UFZ-1H10) . Concluding, the hybrid assembly revealed to be the optimal approach for genome reconstruction, because the advantage of quality (Illumina approach) and read length (Nanopore) and disadvantages of short reads (Illumina) and lower quality (Nanopore) complement each other. Illumina-based sequencing approaches usually lead to highly fragmented MAGs. This means that complex genomes can only rarely be reconstructed. The functional understanding of microbiomes from AD systems could be expanded and more information could be retrieved from metagenomic samples. After all, an approach that uses a dereplicated set of recovered MAGs from hybrid assemblies is likely to improve our ability to reconstruct genomes and can even enable the strain-specific reconstruction of genomes from metagenomic data."
} | 5,571 |
25980407 | PMC4434858 | pmc | 4,283 | {
"abstract": "Background Host-microbe and microbe-microbe interactions are often governed by the complex exchange of metabolites. Such interactions play a key role in determining the way pathogenic and commensal species impact their host and in the assembly of complex microbial communities. Recently, several studies have demonstrated how such interactions are reflected in the organization of the metabolic networks of the interacting species, and introduced various graph theory-based methods to predict host-microbe and microbe-microbe interactions directly from network topology. Using these methods, such studies have revealed evolutionary and ecological processes that shape species interactions and community assembly, highlighting the potential of this reverse-ecology research paradigm. Results NetCooperate is a web-based tool and a software package for determining host-microbe and microbe-microbe cooperative potential. It specifically calculates two previously developed and validated metrics for species interaction: the Biosynthetic Support Score which quantifies the ability of a host species to supply the nutritional requirements of a parasitic or a commensal species, and the Metabolic Complementarity Index which quantifies the complementarity of a pair of microbial organisms’ niches. NetCooperate takes as input a pair of metabolic networks, and returns the pairwise metrics as well as a list of potential syntrophic metabolic compounds. Conclusions The Biosynthetic Support Score and Metabolic Complementarity Index provide insight into host-microbe and microbe-microbe metabolic interactions. NetCooperate determines these interaction indices from metabolic network topology, and can be used for small- or large-scale analyses. NetCooperate is provided as both a web-based tool and an open-source Python module; both are freely available online at http://elbo.gs.washington.edu/software_netcooperate.html.",
"conclusion": "Conclusions Network analysis has become an essential component in the study of microbiology. Metabolic, regulatory, and protein-interaction networks provide insight into the behavior and dynamics of individual cells [ 31 - 34 ], whereas ecological networks reveal processes defining the behavior of entire microbial communities [ 29 , 35 , 36 ]. Yet, molecular network properties are rarely used to explain patterns observed in ecological networks, although clearly, these two scales of organization are tightly linked. The reverse-ecology framework provides a powerful platform to address this challenge and to couple genomic information with environmental context. Specifically, the Biosynthetic Support Score and the Metabolic Complementarity Index represent two successful examples in which molecular network analysis can be applied to ecological studies of microbe-microbe and host-microbe interactions. Unfortunately, the implementation of such graph theory-based methods is not trivial, and may be beyond of the technical capabilities of microbiology researchers with no advanced computational skills. Above, we have presented NetCooperate, a web-based tool and Python package for easily performing the necessary computation. NetCooperate can be applied on a small-scale by those studying a microbe of interest, or it can be integrated into a larger workflow for large-scale analysis of entire communities. NetCooperate, along with previously introduced methods [ 14 , 17 ], completes the suite of reverse-ecology analysis tools accessible to researchers with any level of technical expertise.",
"discussion": "Results and Discussion We have previously successfully utilized the cooperation metrics calculated by NetCooperate for studying a number of microbial systems and have shown that they provide tools for addressing fundamental questions in microbial ecology and evolution [ 18 , 24 , 27 ]. Such studies demonstrate the benefits of using systems-level tools and the impact such tools can have on elucidating global principles that govern multi-species systems. Specifically, below we discuss two such studies we have conducted that highlight the potential of NetCooperate and its applicability to several systems of interest [ 18 , 24 ]. These studies have promoted much interest and the application of this approach to address various challenges in biotechnological and medical settings has been highlighted [ 11 - 13 ]. It is our hope that providing the NetCooperate tool will enable the research community at large to apply this framework to a wide array of microbial ecosystems. Predicting host-parasite interaction and characterizing patterns of parasite adaptation Parasitic species are clearly well adapted to their hosts. In introducing the Biosynthetic Support Score, Borenstein and Feldman aimed to examine whether such adaptation is reflected in the species’ metabolic networks and whether it can be used to predict parasitic species and specific host-parasite interactions [ 24 ]. To this end, they used the Biosynthetic Support Score to quantify the interaction between approximately 600 bacterial species and each of three model eukaryotic hosts ( human , fruit fly , and Arabidopsis , representing a mammalian, insect, and plant host respectively). The distribution of BSS values of all bacteria against all hosts ranged from approximately 0.45 to 0.95. Importantly, a comparison of pathogenic bacteria to free living bacteria showed that parasitic bacteria have significantly higher BSS with all three hosts compared to free-living bacteria and that BSS was better in predicting parasitic species than classical metrics (e.g., genome size). Moreover, the BSS of a given parasite was higher when the model host was phylogenetically related to the parasite’s natural host (e.g., mammalian parasites had significantly greater BSS in human than in fruit fly), suggesting that the parasite’s metabolic network was sufficient to infer not only its parasitic life-style but also its preferred host. To demonstrate the applicability of the Biosynthetic Support Score to evolutionary analysis, this study further integrated this cooperation score with phylogenetic analysis, calculating the BSS of both extant and ancestral species (obtained through phylogenetic reconstruction) within the phylum Firmicutes . It was then shown that the biosynthetic support provided by human to any given bacterium increased with the phylogenetic distance of the species from the common ancestor of Firmicutes, clearly demonstrating the gradual adaptation of parasites to their host environment on a global scale. Given the success of the BSS metric in predicting host-parasite interactions, it was later also proposed as a tool for designing culture media and for studying host-microbiome interactions [ 12 , 28 ]. Assessing interaction between co-occurring microbes and elucidating assembly rules in the human microbiome The human microbiome is a diverse and complex microbial ecosystem, with different individuals harboring markedly different sets of species. Previous surveys of the microbiome have revealed clear non-neutral patterns in the distribution of species and have demonstrated that certain species pairs tend to co-occur across microbiome samples whereas others tend to exclude one another [ 29 ]. Yet, the underlying forces that give rise to these patterns were not clear. The Metabolic Complementarity Index was first developed to address this challenge and to study emergent organizational properties of community assembly in the human microbiome [ 18 ]. This metric was first validated by predicting metabolic complementarity among several species of the human oral microbiota with well-characterized and assayed interactions [ 30 ], to confirm that it correctly identified preferred interacting partners. Indeed, in a series of controlled in vitro experiments, where microbes were placed in a nutrient-limited saliva medium, microbes were found to grow best in the presence of species with greater metabolic complementarity. In such settings, the ability of species to complement the nutritional requirements of their partners translates into active cooperation and improved growth. Moving on to in vivo communities of the human intestine, the MCI between all possible pairs among >150 gut dwelling microbial species was then calculated. By comparing the MCI among species’ co-occurring partners to their excluders , it was found that in fact in this nutrient-rich environment species with low MCI tended to co-occur, whereas species pairs with greater MCI excluded one another from a given host-habitat. This finding suggested that in the assembly of these communities habitat filtering outweighed the impact of species interaction and that species relied on the availability of nutrients in the environment rather than realizing the potential for cross-feeding [ 18 , 27 ]. Put differently, in this nutrient-rich environment, the potential for cooperation did not necessarily materialize and species assortment was based on the availability of nutrients in the environment rather than on the presence or absence of other species. An in depth analysis further revealed that not only is MCI not an artifact of phylogenetic relatedness, but that it was more successful at predicting species interactions. A similar analysis was used to investigate community assembly across multiple phylogenetic and biogeographic scales, demonstrating that metabolic complementarity had a greater influence on species co-occurrence patterns between members of the same phylum than across all species. Finally, applying the MCI to species co-occurrence across and within multiple body sites revealed that habitat filtering is a general assembly rule applicable to communities inhabiting heterogeneous anatomical sites within the human body."
} | 2,440 |
30034526 | PMC6052542 | pmc | 4,287 | {
"abstract": "Background Lignocellulosic biomass is seen as an abundant renewable source of liquid fuels and chemicals that are currently derived from petroleum. When lignocellulosic biomass is used for ethanol production, the resulting liquid residue (stillage) contains large amounts of organic material that could be further transformed into recoverable bioproducts, thus enhancing the economics of the biorefinery. Results Here we test the hypothesis that a bacterial community could transform the organics in stillage into valuable bioproducts. We demonstrate the ability of this microbiome to convert stillage organics into medium-chain fatty acids (MCFAs), identify the predominant community members, and perform a technoeconomic analysis of recovering MCFAs as co-products of ethanol production. Steady-state operation of a stillage-fed bioreactor showed that 18% of the organic matter in stillage was converted to MCFAs. Xylose and complex carbohydrates were the primary substrates transformed. During the MCFA production period, the five major genera represented more than 95% of the community, including Lactobacillus , Roseburia , Atopobium , Olsenella , and Pseudoramibacter . To assess the potential benefits of producing MCFAs from stillage, we modeled the economics of ethanol and MCFA co-production, at MCFA productivities observed during reactor operation. Conclusions The analysis predicts that production of MCFAs, ethanol, and electricity could reduce the minimum ethanol selling price from $2.15 to $1.76 gal −1 ($2.68 gal −1 gasoline equivalents) when compared to a lignocellulosic biorefinery that produces only ethanol and electricity. Electronic supplementary material The online version of this article (10.1186/s13068-018-1193-x) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion In this study, we tested the hypothesis that microbial communities could be used to produce valuable compounds from lignocellulosic stillage. We developed conditions for sustained MCFA production by an anaerobic microbiome that uses stillage produced during lignocellulosic biorefining. By fermenting switchgrass stillage, we maintained productivities of hexanoic and octanoic acids of 2.6 ± 0.3 and 0.27 ± 0.04 g L −1 day −1 , respectively. To our knowledge, this is the first demonstration of MCFA production with xylose and other organics in lignocellulosic ethanol stillage as the primary substrates. The MCFA-producing microbial community was derived from a diverse wastewater treatment ecosystem, but over time it became enriched with OTUs representing only five genera, including members of the Firmicutes phylum ( Lactobacillus , Roseburia , and Pseudoramibacter ) and of the Actinobacteria phylum ( Olsenella and Atopobium ). Pseudoramibacter are Clostridia related to known MCFA-producing organisms, some of which have been shown to produce hexanoic and octanoic acids [ 66 ]. A TEA, based on an update to an industry-accepted model, shows that, at the productivity of MCFAs achieved in this study, valorizing lignocellulosic ethanol stillage to MCFAs could improve the economic sustainability of a biorefinery. For example, using the MCFA production experimentally observed, if 16% of the COD remaining in stillage is converted to hexanoic acid and 1.7% is converted to octanoic acid, the minimum ethanol selling price could be reduced by 18%, from $2.15 to $1.76 gal −1 . Optimization of microbiome MCFA productivities, MCFA extraction, solvent recovery and selection of the ethanologenic organism may contribute further to improving the economy of the lignocellulosic biorefinery.",
"discussion": "Discussion Our work illustrates the potential of using microbial communities to convert stillage into valuable co-products. In the stillage-fed bioreactor, productivities of hexanoic (2.6 ± 0.3 g L −1 day −1 ) and octanoic (0.27 ± 0.04 g L −1 day −1 ) acids were sustained for 214 days with titers at 66 ± 8.2 and 97 ± 15% of their solubility in water, respectively. These productivities are consistent with other studies investigating the conversion of organic substrates derived from lignocellulosic materials or ethanol production wastes to MCFAs (Additional file 8 : Table S17). Our system is unique, however, in that the primary carbohydrate consumed is xylose and the stillage has already been depleted of a large portion of fermentable sugars and the ethanol that others have used to produce MFCA. While we are proposing the co-production of ethanol and MCFAs in this study, recent work has also explored production of MCFA as the main product of a lignocellulosic biorefinery. In work performed by Nelson et al., Megasphaera consumed glucose in lignocellulosic hydrolysate to generate hexanoic acid, but xylose was not consumed [ 16 ]. The microbial community like the one presented in this study could be utilized to convert the remaining xylose to MCFAs. The simplicity of the microbial community enriched in this study positions it well as a model community for MCFA production. Others have shown enrichments containing OTUs related to primary sugar fermenters, such as Lactobacillus , and OTUs related to Clostridia that may be involved in converting intermediate fermentation products to MCFA [ 13 – 15 , 17 , 18 , 47 , 69 ]. In our microbial community, at Day 252, only 10 OTUs are present at greater than 1% relative abundance, and these OTUs make up 89.3% of the total OTUs (Additional file 5 : Figure S3). The statistical analyses indicate that Pseudoramibacter and Lactobacillus are co-enriched, and their abundance correlates with higher MCFA production. We, therefore, propose that Lactobacillus converts xylose to lactate and acetate by heterofermentation, and the lactate is elongated to MCFAs by Pseudoramibacter . While 16S rRNA gene sequencing allows for the phylogeny of abundant organisms to be estimated, the function of community members should be investigated further utilizing metagenomic approaches. Due to the simplicity of the microbial community obtained in this study, this microbiome is well positioned for further investigation with metagenomic tools. Furthermore, its simplicity makes this a candidate microbiome for simulation with synthetic communities in the future. Of the OTUs that became enriched in the reactor, only Roseburia (denovo27808) and Pseudoramibacter (denovo6337) emerged as likely MCFA-producing bacteria. While Pseudoramibacter have been shown to produce MCFAs [ 66 ], to our knowledge, the ability of Roseburia to produce MCFAs has not been studied. The TEA shows that even at the modest productivities of hexanoic and octanoic acids obtained in this study, MCFAs produced from ethanol stillage could improve the economic feasibility of lignocellulosic biorefining if the productivity can be maintained at industrial scale. Improvements in the overall conversion of stillage COD to MCFAs and production of a higher proportion of octanoic acid would further increase the revenue that can be generated by this strategy. Increasing MCFA product specificity towards octanoic acid is an ongoing area of research. One strategy to increase octanoic acid production is to utilize pertractive extraction of MCFAs to reduce product inhibition, as has been performed in past studies [ 13 , 14 ]. Recent work has also shown that increasing the ratio of ethanol to acetate increases selectivity of octanoic acid production [ 13 ]. The model of increasing the ratio of reduced electron donors to acetate suggests that, in the absence of ethanol, increasing the production of lactate as a fermentation intermediate (rather than acetate) could further drive octanoic acid production. The economy of co-producing MCFAs may also be affected by upstream biomass processing (i.e., the conversion of plant polymers to their constituent monomer units) and ethanol fermentation. For example, utilization of xylose by industrial yeast strains, such as S. cerevisiae , is limited [ 20 ], although attempts to improve pentose utilization by ethanol producers is an area of intense research activity [ 70 ]. Even though the S. cerevisiae Y128 strain used in this study was engineered for improved xylose utilization, it only consumed 47% of the xylose available in the switchgrass hydrolysate. Future ethanologenic organisms used in a lignocellulosic biorefinery may leave less xylose available for MCFA production. However, given the higher price of MCFAs compared to ethanol, decreasing xylose consumption by the ethanologenic organism may actually result in an improved economy of the lignocellulosic biorefinery. Another simple opportunity for improving the economic potential of co-producing MCFAs is utilizing sodium hydroxide for pH control, instead of KOH, as sodium hydroxide is roughly one-sixth the cost of KOH. In our current model, the cost of KOH (Table 3 ) is a major expense. Alternatives to controlling pH with chemicals, such as electrolytic extraction which both controls the pH and extracts the acid products [ 17 ], should also be explored further."
} | 2,266 |
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