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39512191 | PMC11632650 | pmc | 6,150 | {
"abstract": "Under 3D culture conditions, cells tend to spread, migrate,\nand\nproliferate better in more viscoelastic and plastic hydrogels. Here,\nwe present evidence that the improved cell behavior is facilitated\nby the lower steric hindrance of a more viscoelastic and plastic matrix\nwith weaker intermolecular bonds. To determine intermolecular bond\nstability, we slowly insert semispherical tipped needles (100–700\nμm diameter) into alginate dialdehyde-gelatin hydrogels and\nmeasure stiffness, yield strength, plasticity, and the force at which\nthe surface ruptures (puncture force). To tune these material properties\nwithout affecting matrix stiffness, we precross-link the hydrogels\nwith CaCl 2 droplets prior to mixing in NIH/3T3 fibroblasts\nand final cross-linking with CaCl 2 . Precross-linking introduces\nmicroscopic weak spots in the hydrogel, increases plasticity, and\ndecreases puncture force and yield strength. Fibroblasts spread and\nmigrate better in precross-linked hydrogels, demonstrating that intermolecular\nbond stability is a critical determinant of cell behavior under 3D\nculture conditions.",
"conclusion": "Conclusions Our study introduces a novel metric, the\nsurface puncture force,\nto estimate the steric hindrance encountered by cells embedded in\na hydrogel. We showed that precross-linked ADA-GEL hydrogels exhibited\nsignificantly lower puncture forces, lower yield strength, and higher\nplasticity compared to control ADA-GEL hydrogels while at the same\ntime exhibiting improved cell proliferation, a higher degree of spreading,\nand a higher proportion and speed of migrating cells. This finding\nsuggests that plasticity, and in particular surface puncture force,\nwhich is a readily measurable parameter, predicts cell behaviors that\nare predominantly influenced by the steric hindrance of a 3D matrix.\nOur finding may help accelerate the development of novel bioinks with\nimproved cell biocompatibility.",
"introduction": "Introduction Bioprinting is a method of biofabrication\nin which living cells\nare suspended in a highly viscous or pasty matrix, forming a bioink.\nAfter extrusion of the bioink through the nozzle of a 3D printer,\nfollowed by cross-linking or polymerization for mechanical fixation,\nthe resulting biofabricate should have appropriate structural, mechanical,\nand adhesive properties to provide a biocompatible environment for\ncells. One of the key challenges is to optimize and tailor the physicochemical\nproperties of the biofabricate to achieve desired cell behavior, such\nas cell proliferation and colonization of the matrix, cell differentiation,\ncell migration, and endothelialization. This requires a thorough understanding\nof the interactions between the cells and bioprinted material. A widely used and versatile class of bioinks with tunable properties\nare alginate-based hydrogels. 1 Alginate\nis a biocompatible natural polysaccharide. It is inexpensive, has\ngood printability and extrusion fidelity, is largely bioinert and\nnonadhesive, and can be cell-friendly cross-linked with Ca 2+ ions. Alginate can be bioprinted as-is, or it can be oxidized to\nalginate dialdehyde (ADA), whereby aldehyde groups along the polysaccharide\nbackbone are used as linkers for further chemical modification. 2 For example, amino groups of cell adhesion proteins\nsuch as collagen or laminin can be covalently attached to the aldehyde\ngroups through a Schiff’s base reaction by simply mixing the\ndesired protein with ADA prior to cross-linking. To further improve\nthe biocompatibility and degradation properties of ADA, high concentrations\n(2–5 wt %) of gelatin (GEL) can be added to the mixture, followed\nby cross-linking with transglutaminase, to form an ADA-GEL copolymer. 3 − 5 Gelatin further improves the adhesion properties of the bioink,\nis far less expensive compared to purified extracellular matrix proteins,\nand is stable at room temperature. When cells such as dermal\nfibroblasts are seeded on top of ADA-GEL\nhydrogel surfaces, they readily attach, spread, and proliferate. 5 By contrast, when the cells are mixed into the\nADA-GEL matrix, we found that they spread and proliferate poorly,\nalthough they remain viable. 6 Precross-linking\nthe ADA-GEL bioink with low to moderate concentrations of Ca 2+ ions prior to extrusion and post cross-linking greatly improved\ncell migration, spreading, and proliferation. 6 Precross-linked ADA-GEL hydrogels were similarly stiff compared\nto non-precross-linked ADA-GEL hydrogels but displayed a more viscoelastic\nbehavior. 6 This finding was in agreement\nwith previous reports claiming that increasing the viscous behavior\n(or equivalently the stress relaxation time constant) of otherwise\nsimilarly stiff viscoelastic hydrogels improved cell biocompatibility. 7 , 8 Furthermore, precross-linked ADA-Gel is one of the few extrusion-printable\nbioinks that support rapid cell proliferation and invasion/migration\ncomparable to nonprintable biopolymer hydrogels (e.g., Matrigel, collagen). We discovered that with increasing degree of precross-linking,\nthe ADA-GEL hydrogels became not only more viscoelastic but also more\nplastic, as demonstrated by the degree of stress relaxation after\napplying a 5% compressive strain for 10 min. Changes in cell proliferation,\nspreading, and migration were more closely correlated with hydrogel\nplasticity than with the viscoelastic relaxation time constant. This\nis consistent with other reports that have found that different patterns\nof plastic remodeling regulate biological outcomes such as fibroblast\nactivation, 9 cancer cell migration, 10 and vascular assembly and invasion. 11 In the present work, plasticity refers to the\ninelastic, irreversible matrix deformations that result from the mechanical\nbreakage of intermolecular bonds. Since alginate-based hydrogels are\nnot degraded by cell-secreted proteases, 12 cells must mechanically break matrix bonds by force application\nin order to spread, migrate, and proliferate. Therefore, we reasoned\nthat plasticity may be a reliable predictor of cell behavior because\nit reports the mechanical stability of the material’s intermolecular\nbonds. Here, we provide more direct evidence that precross-linking\nof\nADA-GEL hydrogels reduces the force at which intermolecular bonds\nin the hydrogel break. To demonstrate this, we puncture the surface\nof ADA-GEL hydrogels with blunt needles and record the force at which\nthe hydrogel surface ruptures and the needle penetrates the 3D matrix,\nin addition to confirming with new precross-linking methods that increased\nplasticity correlates to biological behavior. We then correlate the\npuncture forces of different hydrogels with the spreading and migration\nbehaviors of cells grown in these hydrogels. We conclude that puncture\nforce experiments provide a simple and novel method to estimate the\nprotrusive cell forces required for spreading and migration and may\naid in the design of novel bioinks for improved cell behavior.",
"discussion": "Results and Discussion Results : Precross-linking ADA-GEL with Ca 2+ ions results in similar mechanical properties but changes their\nmicrostructure. We modified a previously established method\nfor precross-linking ADA-GEL with Ca 2+ ions to achieve\nimproved optical clarity: instead of mixing ADA-GEL with CaCO 3 particles, 6 we directed an ultrasound-nebulized\nstream of CaCl 2 solution droplets onto an ADA-GEL mixture.\nIn order to homogenize the dispersion of droplets, the mixture was\ncontinuously stirred during the nebulization process ( Figure 1 a). The nebulization time was\nadjusted to obtain a lower (40 mM) or higher (60 mM) total concentration\nof CaCl 2 in the mixture. A control condition is obtained\nby nebulizing water into the mixture. As a final step, the ADA-GEL\nmixtures were enzymatically and ionically cross-linked with transglutaminase\n(4 mg/mL) and 100 mM CaCl 2 in distilled water (see Experimental\nSection (Materials and Methods)). The precross-linked ADA-GEL showed\na heterogeneous structure with differently reflective regions and\nspots when observed through a confocal reflection microscope ( Figure 1 b,c). The size of\nthese reflective spots increased with the degree of precross-linking\n( Figure 1 b). From the\nobservation that alginate hydrogels scatter more light with an increasing\ndegree of cross-linking, we conclude that precross-linking with CaCl 2 droplets introduces a separation of the hydrogel into regions\nwith a higher or lower degree of ADA-cross-linking. We prepared\nADA-GEL hydrogels with FITC-stained gelatin to measure\nthe homogeneity of the gelatin phase. We then compared the confocal\nreflectance and fluorescence signals of the same region ( Figure 1 c). We found that\nfew hydrogel regions have both high reflectance and fluorescence,\nbut most other regions showed no clear colocalization of the reflectance\nand fluorescence signals. In particular, smaller spots were mostly\nseen in the reflectance channel but not in the fluorescence channel.\nFrom this, we conclude that precross-linking introduces regionally\nuncorrelated inhomogeneities in both the degree of cross-linking and\nthe concentration of gelatin. Next, we investigated the macroscopic\nmechanical behavior of ADA-GEL\nthat was not precross-linked, pre-crosslinked with 40 mM CaCl 2 and 60 mM CaCl 2 . First, the swelling and degradation\nproperties of the ADA-GEL hydrogels were analyzed by measuring the\nspecific weight w of the three hydrogel formulations\nover 25 days. As shown in Figure 2 a, the swelling ratio of ADA-GEL hydrogels (the ratio w ( t ) /w ( t = 0)) rapidly increased during the first 6 h after immersion in\nDMEM and then decreased slowly over the following days but decreased\nmore rapidly after day 18. The peak swelling ratio measured after\n6 h was highest—about 50%—for the control (nonprecross-linked)\nADA-GEL and lower—about 20–30%—for the precross-linked\ngel. The swelling ratio followed similar trends in all three hydrogel\nformulations. Next, we measured the mechanical properties of\nthe hydrogels using\nmicroindentation with a 3 mm diameter indenter. These measurements,\ncarried out over a period of 18 days, showed that the stiffness (Young’s\nmodulus) was similar for all conditions ( Figure 2 b). Young’s moduli were obtained through\nlinear fitting of the force–indentation curves in the unloading\nphase ( Figure 2 c and\nExperimental Section (Materials and Methods)). Stiffness was highest\nimmediately after final cross-linking (at day 0, 3.6 kPa for the control\nsamples and 3.1 kPa for both precross-linked samples) and decreased\nsimilarly for all conditions over time when immersed in DMEM, regardless\nof their degree of precross-linking. Given the importance of\nalginate-based hydrogels as bioinks in\ntissue engineering, we also investigated the extent to which extrusion\nstability is dependent on the degree of precross-linking. Figure 2 d shows that the\nextrusion stability was better at 23 °C compared to that at 37\n°C. In addition, the precross-linked conditions showed slightly\nimproved extrusion stability at 23 and 37 °C compared to the\ncontrol group. With a 200 μm printing capillary, the print resolution\nat 23 °C was 0.5 mm for the 40 and 60 mM conditions and 0.6 mm\nfor the control condition, while at 37 °C, the print precision\nwas 0.7 mm for the 40 and 60 mM conditions and 0.8 mm for the control\ngroup. Precross-Linking ADA-GEL Improves Cellular Behavior We evaluated the biological properties of ADA-GEL precross-linking\nby performing morphological, proliferation, and migration assays of\nNIH-3T3 cells 3D-embedded in the gel. We used a FUCCI-based NIH-3T3\ncell line as a proliferation reporter ( Figure 3 a). In this cell line, two proteins that\nare reciprocally active during the cell cycle are labeled with different\nfluorescent proteins so that the fluorophore visible at the nucleus\nindicates the phase of the cell cycle. Figure 3 b shows that proliferation changed as a function\nof both time and the degree of precross-linking. The percentage of\nproliferative cells was considerably higher in the 40 and 60 mM precross-linked\nADA-GEL hydrogels compared to that in control conditions. From day\n3 to day 7, the percentage of proliferative cells decreased under\nall conditions but remained highest in 60 mM precross-linked ADA-GEL\nhydrogels. Next, we evaluated the morphology of NIH-3T3 tdTomato\ncells embedded in the hydrogels as a function of both precross-linking\nand time in culture ( Figures 3 c,d and S1, Supporting Information ). We measured circularity and aspect ratio (major axis divided by\nthe minor axis) as two inversely related morphological parameters.\nCells spreaded similarly in the two precross-linked ADA-GEL conditions\nas early as 1 day after embedding, whereas the cells maintained a\nround morphology in the nonpre-cross-linked control hydrogels. After\nday 1, cell morphology remained stable over time up to 7 days after\nembedding. We then measured the ability of NIH-3T3 tdTomato\ncells to migrate\nwithin the ADA-GEL hydrogels depending on the degree of precross-linking\n( Figure 4 ). Consistent\nwith the increased proliferation and spreading in precross-linked\nADA-GEL mixtures, the cell motile fraction and migration speed were\nhigher in the precross-linked ADA-GEL compared to that in control,\nboth at 3 and 6 days in culture. These differences were already manifested\nafter 24 h of culture (Figure S2, Supporting Information ). For all metrics except motile fraction, the two precross-linked\nconditions (with 40 and 60 mM CaCl 2 ) were not significantly\ndifferent from each other but showed clearly significantly different\nvalues compared to control. Motile fractions increased significantly\nfrom control to the highest degree of precross-linking at day 3, but\nnot at day 7, when the intermediate precross-linked condition was\nsimilar to control. Cell viability over 1 week of culture was similarly\nhigh for both control precross-linked samples (Figure S3, Supporting Information ). Figure 4 Cell migratory behavior\nfor ADA-GEL with different degrees of precross-linking.\n(a) 24 h migration (mean and standard error over individual field\nof views, with an average of ∼800 cells tracked) of NIH/3T3\ncells recorded starting at day 3 in terms of persistence, mean displacement,\nmotile fraction, and speed for the ADA-GEL control, ADA-GEL precross-linked\nwith 40 mM CaCl 2 , and ADA-GEL precross-linked with 60\nmM CaCl 2 . (b) 24 h migration of NIH/3T3 cells recorded\nstarting at day 6, (c) representative trajectories of all cells tracked\nover 12 h within a field of view in the ADA-GEL control, ADA-GEL precross-linked\nwith 40 mM CaCl 2 , and ADA-GEL precross-linked with 60\nmM CaCl 2 . The blue colored trajectory highlighted by the\ndotted box corresponds to the trajectory in (d). (d) Representative\nminimum intensity-projection brightfield images and trajectories of\nan individual NIH/3T3 cell migrating through the ADA-GEL precross-linked\nwith 60 mM CaCl 2 . Statistical differences between different\nADA-GEL were tested using ANOVA (* = p < 0.05,\n** = p < 0.005, *** = p <\n0.0005, n.s = p > 0.05). Scale bar = 200 μm. Precross-Linked ADA-GEL Have Lower Puncture Force and Yield\nStrength and Higher Plasticity To test whether the enhanced\ncellular behavior in precross-linked ADA-GEL was due to a lower bond\nstrength of the matrix, we penetrated the material with blunt (semispherical\ntipped) needles with diameters of 100, 300, and 700 μm and recorded\nthe corresponding force–depth profiles ( Figure 5 a,b). For all diameters, we found a characteristic force–depth\nprofile ( Figure 5 b)\nwhere the force first slowly increased and then suddenly dropped.\nConcurrent microscopic images confirmed that the slow force increase\nwas associated with a downward bending of the hydrogel surface and\nthe sudden force decrease with a penetration of the needle through\nthe hydrogel surface into the hydrogel whereby the surface recoiled\nupward ( Supporting Information Videos 1–3).\nFollowing the established nomenclature, we refer to the force value\nimmediately before the sudden drop as the “puncture force”,\nwhich is the force required to penetrate the gel surface. The subsequent\nforce–depth profile after surface puncture was more irregular\nfor precross-linked ADA-GEL hydrogels ( Figure 5 b). Figure 5 Cell–material interactions and puncture\nexperiments to link\nto increased migration/spreading. (a) Experimental setup: a cylindrical\nsteel needle with a hemispherical tip with diameter D was driven with a defined velocity into a ADA-GEL hydrogel. The\nindentation force was monitored with a standard laboratory precision\nscale, (b) representative force versus indentation curves measured\nwith a 300 μm diameter hemispherical needle for ADA-GEL control,\nADA-GEL precross-linked with 40 mM CaCl 2 , and ADA-GEL precross-linked\nwith 60 mM CaCl 2 . Arrows indicate the point when the surface\nis punctured. (c) Surface puncture forces from n =\n15 samples per condition and needle plotted as a function of the hemispherical\ntip diameter for the three conditions as boxplots indicating the median,\nthe 25th and 75th percentiles (box), and the most extreme data points\nnot considered outliers (whiskers). Statistical differences between\ndifferent ADA-GEL were tested using ANOVA (* = p <\n0.05, ** = p < 0.005, *** = p < 0.0005, n.s = p > 0.05). Importantly, the puncture force for precross-linked\nADA-GEL hydrogels\nwas 1 order of magnitude lower than for nonprecross-linked ADA-GEL\nat all diameters ( Figure 5 c). There was no statistically significant difference between\nthe puncture forces of the low (40 mM) and high (60 mM) precross-linked\nconditions. To further validate our hypothesis that the improved\ncell behavior\nat higher degrees of cross-linking is due to a lower yield strength\nand higher plasticity of the hydrogels, we additionally used a different\nprecross-linking method, which allowed us to add four finely tuned\ndegrees of precross-linking to the control ADA-GEL, i.e., 20, 40,\n60, and 80 mM CaCl 2 . The hydrogels were punctured using\na 300 μm-diameter, hemispherical-tipped puncture needle, which\nwas repeatedly inserted into the material and then withdrawn at progressively\ngreater depths of penetration, typically 3 to 4 mm ( Figure 6 a–c). This protocol allowed us to measure Young’s\nmodulus (for small indentation depths), viscoelastic behavior, yield\nstrength, and plastic deformation within the same experiment after\n3 days in culture with embedded cells. We first confirmed that the\nYoung’s modulus, calculated using the Hertz theory, was unaffected\nby the degree of precross-linking. Conversely, viscoelastic relaxation,\nquantified by three parameters from a stretched exponential fit of\nstress relaxation over 300 s, was dependent on precross-linking, in\nparticular the relaxation time constant. When we quantified plastic\nyield as residual deformation versus maximum indentation, we found\nan increase with the degree of precross-linking ( Figures 6 b,c and S4–S6, Supporting Information ). We further quantified\nthe spreading of NIH-3T3 tdTomato cells embedded in the same hydrogels\nafter 3 days in culture using the circularity metric and confirmed\nan increase in spreading with the degree of precross-linking (Figure\nS7, Supporting Information ). Taken together,\nwe found no correlation between the elastic response (Young’s\nmodulus) and the biological behavior (spreading) ( Figure 6 d) and stronger correlations\nwith the viscoplastic response ( Figure 6 e,f). Figure 6 Elasto-viscoplastic profiles of precross-linked ADA-GEL\nhydrogels\nand their relation to cell spreading after 3 days in culture. (a)\nExperimental indentation setup: a cylindrical steel needle with a\nhemispherical tip of 300 μm diameter was driven into an ADA-GEL\nhydrogel with a defined velocity profile. The indentation force was\nmonitored using a standard laboratory precision balance. The history\nof the velocity profile was designed to repeatedly insert and withdraw\nthe tip into the hydrogel at progressively larger indentation depths\nto determine the plastic deformation and also to obtain the Hertz-calculated\nYoung’s modulus (in three cycles with small indentation depth,\ni.e., 500, 600, 700 μm, shown) and the viscoelastic relaxation\nfit by keeping the indentation constant for 300 s, (b) representative\nprofiles for the ADA-GEL control case, showing the weight vs indentation\ncurves as the indenter was inserted and withdrawn according to the\nindentation history shown in (a) (top left); the residual deformation\nat a threshold force (corresponding to a minimum 50 mg weight threshold)\nversus the maximum indentation for the specific cycles is colored\nas in the previous weight vs indentation curves (top right); the Young’s\nmodulus calculated by Hertz for cycles 5, 6, and 7 (bottom left),\nand the viscoelastic fit using a stretched exponential law (bottom\nright). (c) Representative profiles for the ADA-GEL precross-linked\nwith 80 mM CaCl 2 [see description of the plots in (b)].\n(d–f) Correlations of elasto-viscoplastic parameters (Young’s\nmodulus, residual deformation at 2 mm maximum indentation depth, and\nrelaxation time constant of the viscoelastic fit, respectively) with\ncell spreading (circularity). Each point represents the mean value\nfor a condition (control, 20, 40, 60, and 80 mM precross-linking strength)\nin which both cell spreading and elasto-viscoplastic parameters were\nmeasured. The bars indicate the standard error of the mean for n = 3 replicates of the same measurement. Discussion : In this study, we investigated the relationship\nbetween hydrogel penetration force and the ability of embedded fibroblasts\nto spread, migrate, and proliferate. We compared alginate-based hydrogels\nwith varying degrees of polymer chain precross-linking. Precross-linking\nwas achieved by either nebulizing water droplets with different concentrations\nof Ca 2+ ions onto the surface of a constantly stirred alginate\nsolution or by slow addition of the CaCl 2 precross-linking\nsolution under vigorous stirring prior to mixing-in the cells and\nfinal cross-linking with a high (100 mM) concentration of Ca 2+ ions. Precross-linking did not markedly change the Young’s\nmodulus of the hydrogel after final cross-linking, and the hydrogels\nwere stable over a 10 day period without significant degradation. A previous report showed that precross-linking enhances the proliferation,\nspreading, and migration of embedded cells, and that the enhanced\ncell behavior correlates more strongly with the plasticity of the\nhydrogels and not so much with the viscoelastic relaxation time. 6 The authors of that study speculated that the\nhigher plasticity of the precross-linked hydrogels was attributable\nto a lower intermolecular bond strength of the cross-links, and that\na lower bond strength allowed the cells to overcome the steric hindrance\nof the hydrogels more easily by employing cell-generated protrusive\nand traction forces. In the present study, we set out to test this\nidea by measuring the forces required to penetrate a blunt needle\nthrough the hydrogel surface as a more direct measure of the bond\nstrength. To further investigate how intermolecular bonds are affected\nby the loading rate, we tested nonprecross-linked samples at indentation\nspeeds of 50 and 500 μm/s and observed a slight decrease in\npuncture force at the higher loading rate (Figure S9, Supporting Information ). This result suggests\nthat viscoelastic or viscoplastic deformations stabilize the material\nat lower loading rates, allowing for a time-dependent reorganization\nof molecular chains that counteracts any thermally induced bond rupture\ndynamics, which typically decreases the rupture force at lower loading\nrates. Precross-linking resulted in a heterogeneous hydrogel\nstructure.\nConfocal reflection imaging in precross-linked ADA-GEL hydrogels revealed\nirregular spots and patches of several micrometers in diameter with\nincreased light scattering and thus higher reflection intensities.\nWith increased precross-linking, the number, size, and reflection\nsignal intensity of these structures increased. Since ADA-GEL hydrogels\nscatter visible light when cross-linked, we speculate that these structures\nrepresent regions within the hydrogel with locally higher degree of\ncross-linking,\nand that these regions likely form when nebulized calcium chloride\nsolution droplets come in contact with the bioink during the precross-linking\nstep. As a result, the hydrogel is separated into phases of stronger\nand weaker cross-linking, which can create mechanical weak spots between\nthe phase boundaries, allowing cells to spread and migrate. Alternatively,\nit is also conceivable that cells spread and migrate along or within\nregions with a lower degree of cross-linking; 7 , 21 however,\nwe did not observe a higher occupancy of cells in regions with lower\nreflection signal intensities and hence lower degree of cross-linking. Spreading and migration require the cell to overcome the steric\nhindrance of the extracellular matrix. In amorphous, nonporous (relative\nto the size of cells and cell protrusions) materials, spreading and\nmigration inevitably require the cells to break matrix bonds through\nthe generation of mechanical forces. 22 , 23 The forces\nneeded to break molecular bonds in a hydrogel matrix can be estimated\nfrom the surface puncture force. 15 , 24 Hence, we\nhypothesize that cell spreading and migration increase in hydrogels\nwith a lower puncture force. The study’s primary result\nconfirms this hypothesis: cells\ndisplayed enhanced elongation, migration, and proliferation within\nprecross-linked hydrogels, which had a much lower puncture force compared\nto control hydrogels ( Figures 5 and S10, Supporting Information ). In addition, we developed a new precross-linking protocol involving\nvigorous stirring, which allowed us to more homogeneously distribute\nthe precross-linking throughout the hydrogel. In these homogeneously\nprecross-linked hydrogels, we confirmed that the improved cell behavior\nat higher degrees of precross-linking was due to the lower yield strength\nand higher plasticity of the hydrogels ( Figures 6 and S4–S6, Supporting Information ). This also holds true for the highest precross-linking\nconcentration of 80 mM CaCl 2 , where the viscoelastic and\nplastic material properties of the hydrogels reversed toward those\nseen in nonpre-cross-linked hydrogels ( Figure 6 d), as did the cell spreading behavior (Figure\nS4, Supporting Information ). Our\ndata are in support of a previous study where we showed that\ncell behavior in differently cross-linked hydrogels correlates not\nso much with the viscoelastic stress relaxation time constant 25 but instead with hydrogel plasticity, as measured\nby the nonrecoverable stress amplitude after applying a compressive\nstrain of 5%. 6 For an amorphous,\nnonporous (at the micrometer-scale) bioink such\nas ADA-GEL, our data show that the puncture force of a smooth surface\nis considerably higher than the penetration force after the surface\nhas ruptured, and cracks have likely formed within the material. We\npropose that a cell embedded in a biomaterial ink must first generate\nhigh local forces to puncture the surface at the cell–material\ninterface before the cell can elongate and migrate with lower forces.\nThis would explain why we see not only a decrease in cell elongation\nand migration speed in hydrogels with higher puncture force but also\na considerably lower fraction of motile cells, albeit randomly moving\n(as demonstrated by the persistence metric). Accordingly, the nonmotile\ncell fraction represents, at least to some extent, those cells that\nwere unable to generate sufficient forces to rupture the surface at\nthe cell–material interface.\n\nDiscussion : In this study, we investigated the relationship\nbetween hydrogel penetration force and the ability of embedded fibroblasts\nto spread, migrate, and proliferate. We compared alginate-based hydrogels\nwith varying degrees of polymer chain precross-linking. Precross-linking\nwas achieved by either nebulizing water droplets with different concentrations\nof Ca 2+ ions onto the surface of a constantly stirred alginate\nsolution or by slow addition of the CaCl 2 precross-linking\nsolution under vigorous stirring prior to mixing-in the cells and\nfinal cross-linking with a high (100 mM) concentration of Ca 2+ ions. Precross-linking did not markedly change the Young’s\nmodulus of the hydrogel after final cross-linking, and the hydrogels\nwere stable over a 10 day period without significant degradation. A previous report showed that precross-linking enhances the proliferation,\nspreading, and migration of embedded cells, and that the enhanced\ncell behavior correlates more strongly with the plasticity of the\nhydrogels and not so much with the viscoelastic relaxation time. 6 The authors of that study speculated that the\nhigher plasticity of the precross-linked hydrogels was attributable\nto a lower intermolecular bond strength of the cross-links, and that\na lower bond strength allowed the cells to overcome the steric hindrance\nof the hydrogels more easily by employing cell-generated protrusive\nand traction forces. In the present study, we set out to test this\nidea by measuring the forces required to penetrate a blunt needle\nthrough the hydrogel surface as a more direct measure of the bond\nstrength. To further investigate how intermolecular bonds are affected\nby the loading rate, we tested nonprecross-linked samples at indentation\nspeeds of 50 and 500 μm/s and observed a slight decrease in\npuncture force at the higher loading rate (Figure S9, Supporting Information ). This result suggests\nthat viscoelastic or viscoplastic deformations stabilize the material\nat lower loading rates, allowing for a time-dependent reorganization\nof molecular chains that counteracts any thermally induced bond rupture\ndynamics, which typically decreases the rupture force at lower loading\nrates. Precross-linking resulted in a heterogeneous hydrogel\nstructure.\nConfocal reflection imaging in precross-linked ADA-GEL hydrogels revealed\nirregular spots and patches of several micrometers in diameter with\nincreased light scattering and thus higher reflection intensities.\nWith increased precross-linking, the number, size, and reflection\nsignal intensity of these structures increased. Since ADA-GEL hydrogels\nscatter visible light when cross-linked, we speculate that these structures\nrepresent regions within the hydrogel with locally higher degree of\ncross-linking,\nand that these regions likely form when nebulized calcium chloride\nsolution droplets come in contact with the bioink during the precross-linking\nstep. As a result, the hydrogel is separated into phases of stronger\nand weaker cross-linking, which can create mechanical weak spots between\nthe phase boundaries, allowing cells to spread and migrate. Alternatively,\nit is also conceivable that cells spread and migrate along or within\nregions with a lower degree of cross-linking; 7 , 21 however,\nwe did not observe a higher occupancy of cells in regions with lower\nreflection signal intensities and hence lower degree of cross-linking. Spreading and migration require the cell to overcome the steric\nhindrance of the extracellular matrix. In amorphous, nonporous (relative\nto the size of cells and cell protrusions) materials, spreading and\nmigration inevitably require the cells to break matrix bonds through\nthe generation of mechanical forces. 22 , 23 The forces\nneeded to break molecular bonds in a hydrogel matrix can be estimated\nfrom the surface puncture force. 15 , 24 Hence, we\nhypothesize that cell spreading and migration increase in hydrogels\nwith a lower puncture force. The study’s primary result\nconfirms this hypothesis: cells\ndisplayed enhanced elongation, migration, and proliferation within\nprecross-linked hydrogels, which had a much lower puncture force compared\nto control hydrogels ( Figures 5 and S10, Supporting Information ). In addition, we developed a new precross-linking protocol involving\nvigorous stirring, which allowed us to more homogeneously distribute\nthe precross-linking throughout the hydrogel. In these homogeneously\nprecross-linked hydrogels, we confirmed that the improved cell behavior\nat higher degrees of precross-linking was due to the lower yield strength\nand higher plasticity of the hydrogels ( Figures 6 and S4–S6, Supporting Information ). This also holds true for the highest precross-linking\nconcentration of 80 mM CaCl 2 , where the viscoelastic and\nplastic material properties of the hydrogels reversed toward those\nseen in nonpre-cross-linked hydrogels ( Figure 6 d), as did the cell spreading behavior (Figure\nS4, Supporting Information ). Our\ndata are in support of a previous study where we showed that\ncell behavior in differently cross-linked hydrogels correlates not\nso much with the viscoelastic stress relaxation time constant 25 but instead with hydrogel plasticity, as measured\nby the nonrecoverable stress amplitude after applying a compressive\nstrain of 5%. 6 For an amorphous,\nnonporous (at the micrometer-scale) bioink such\nas ADA-GEL, our data show that the puncture force of a smooth surface\nis considerably higher than the penetration force after the surface\nhas ruptured, and cracks have likely formed within the material. We\npropose that a cell embedded in a biomaterial ink must first generate\nhigh local forces to puncture the surface at the cell–material\ninterface before the cell can elongate and migrate with lower forces.\nThis would explain why we see not only a decrease in cell elongation\nand migration speed in hydrogels with higher puncture force but also\na considerably lower fraction of motile cells, albeit randomly moving\n(as demonstrated by the persistence metric). Accordingly, the nonmotile\ncell fraction represents, at least to some extent, those cells that\nwere unable to generate sufficient forces to rupture the surface at\nthe cell–material interface."
} | 8,483 |
36631572 | PMC9834261 | pmc | 6,151 | {
"abstract": "In the present study, the superhydrophobic coating was synthesized by spherical silica nanostructures modified with organosilane compounds for glass surfaces. To optimize the conditions in terms of cost-effectiveness and create a super-hydrophobic coating with a high contact angle, the response surface method of the central composite design (CCD) model was performed for the StÖber method, and the contact angle was defined as the response surface for the model. Tetraethoxysilane (TEOS) was used as a precursor and poly(dimethylsiloxane) (PDMS) was used to modify the surface of a spherical silica nanostructure synthesized by a one-step sol–gel method using a base catalyst. The accuracy of the research was checked by the contact angle measurement test and an angle of 162° was obtained. XRD, FT-IR, EDS, SEM, DLS, and AFM analyzes were performed to investigate the synthesis of silica nanostructure. Chemical resistance was performed in acidic, neutral, and alkaline environments and the contact angles were 127°, 134°, and 90°, respectively, which indicates that the coating created on the surface glass has good chemical resistance in acidic and neutral environments.",
"conclusion": "Conclusions The Design-Expert software was used to provide a superhydrophobic coating to optimize the test conditions to save time and money and obtain the best answer for the test parameters. The central composite design method was used to perform all possible experiments to obtain the best result. Silica nanostructures were synthesized by the sol–gel-hydrothermal method according to the number of experiments and designed values and the rotational coating was used to create a thin layer on the glass. The contact angle of each experiment was examined as the answer by the software and the optimized values of the experimental parameters were used to synthesize the optimized sample. The optimized sample was identified by the relevant analyzes and the following results were obtained: To prove the superhydrophobic coating, the contact angle between the drop and water was examined and an angle of 162° was obtained. The powder obtained from silica sol was examined and according to XRD analysis, it is a large part of the amorphous nanostructure. And according to the FT-IR spectrum, the Si–O–Si bond was detected. Silica nanostructures have a uniform spherical morphology with a particle size between 255 and 396 nm and the thickness of this nanostructure is 1.06 μm. The surface roughness on the glass surface indicates a super-hydrophobic coating. According to SEM images and DLS analysis, the optimized sample has a uniform size distribution and according to the amount of zeta potential obtained, it has a desirable dispersion property. For chemical resistance, the coating was placed in three environments acidic, neutral, and alkaline for 24 h, and according to the contact angle obtained, it can be concluded that the coating supplied superhydrophobic in neutral and acidic environments more than in other environments.",
"introduction": "Introduction Smart coatings are nanomaterials that automatically respond to changes in the environment such as heat, light, humidity, temperature, pressure, and pH. The purpose of designing such coatings for higher performance is to increase product life, and significantly reduce maintenance costs 1 – 7 . Due to the unique properties of nanoscale materials and the growing demand for nanomaterials in sectors such as the medical and automotive industries, research and development on nano-based coatings replace conventional polymer coatings 8 , 9 . Smart coatings are classified based on application, performance, reactivity, level of complexity, and manufacturing methods. Active sensing coatings include corrosion and pressure-sensitive coatings. Flame retardant coatings are penetrating and non-penetrating coatings. Anti-powder and antibacterial coatings are known as activating coatings. Easy-to-clean coatings include self-cleaning and anti-graphite coatings. Smart window coverings are optically active coatings. Other coatings are anti-fingerprint, anti-reflective, anti-freeze, and anti-fog 10 . Ultra-waterproof coatings are an important category of smart coatings that have received a lot of attention due to their properties. These coatings can be used in any of the above coatings due to their unique properties. For example, due to biodegradation, they can be used in self-healing and antibacterial coatings 11 – 14 , due to morphology and size in self-cleaning and anti-corrosion coatings 15 – 19 , and due to their chemical properties in antifreeze and anti-vapor 19 – 21 . Superhydrophobic surfaces are known for two important properties, the first is the surface roughness at the micro and nanoscales and the second is the complex structure. Therefore, synthesis methods such as electrochemical deposition 22 , CVD 23 , layer-by-layer (LBL) deposition 24 , hydrothermal 25 , and sol–gel can 26 be used to develop and fabricate the mentioned properties. The sol–gel method consists of two stages of hydrolysis and condensation. The raw materials used are silane and metal alkoxides. Among the advantages of the sol–gel method are low-temperature synthesis, high purity, precise control of particle size and distribution, and the possibility of making new crystalline and non-crystalline materials 27 , 28 . Rough surfaces can be created with the help of SiO 2 29 , Al 2 O 3 30 , and CuSO 4 31 , and with the help of hydrophobic agents such as poly(dimethylsiloxane) (PDMS) 32 , hexadecyltrimethoxysilane (HDTMS) 33 , surfaces with low surface energy can be made. The purpose of using the response surface (method) is to design an experiment that examines the possibility of a quadratic interaction between the parameters in the experiment. With the help of the method CCD, it is possible to improve, optimize the process, and also to diagnose the problems and weak points of the process, as a result, to design a process resistant to external influences that produce a suitable product 34 . In this research, for the first time, the Design-Expert software and the response surface method of the central composite design model (CCD) were used to synthesize a superhydrophobic coating on the glass surface to optimize the parameters of the Stöber process. The contact angle of the water drop was used as the response surface. The selected parameters include deionized water as a hydrolysis agent, ethanol as a solvent, ammonia as a catalyst, and polydimethylsiloxane as a surface modification agent. In this method, experimental design is done by determining the actual levels and coding levels for each parameter (i.e. + 1 for high levels, zero central levels, and -1 for low levels).",
"discussion": "Results and discussion Design-expert and analysis of results There are two factors for making a super-waterproof coating: rough surface and rough surface correction. The StÖber method is one of the methods for synthesizing silica nanoparticles to make a rough surface. The purpose of designing the experiment is to optimize the parameters of the StÖber method to create a suitable rough surface and a surface modifying agent for the superhydrophobic coating. Finally, TEOS was investigated as a fixed precursor and the effect of deionized water (A), ethanol (B), ammonia (C), and PDMS (D). The central composite design (CCD) method was used for the design. In this method, the experimental design is performed by specifying the actual levels and coding levels for each parameter (i.e. for high levels + 1, central levels zero, and low levels − 1). Actual and coded levels are shown in Table 1 . The experimental design matrix with the levels encoded by the software is shown in Table 2 . The obtained contact angle was entered into the software as a response. The actual and coded performance levels were used to conduct practical tests. Surface (− 1111) (experiment no. 10) with a contact angle of 110.4° was the lowest and surface (0000) (experiment no. 6) with a contact angle of 166.5° was the highest. Table 1 Actual levels and coded reaction parameters. Factors Code and level − 1 0 + 1 A: Distilled water 8 10 12 B: Ethanol 10 12 14 C: Ammonia 6 9 12 D: Polydimethylsiloxane 20 30 40 Table 2 Experimental design matrix with coded surfaces. Run Factors WCA actual A B C D 1 0 − 2 0 0 125.7 2 − 1 1 − 1 − 1 119.2 3 − 1 1 1 − 1 132.9 4 − 1 − 1 1 1 142.4 5 2 0 0 0 130.7 6 0 0 0 0 166.5 7 0 0 0 0 165.5 8 − 1 1 − 1 1 112.7 9 − 1 − 1 − 1 − 1 117.3 10 − 1 1 1 1 110.4 11 − 2 0 0 0 118.5 12 0 0 0 0 164.5 13 0 0 0 0 165.8 14 0 2 0 0 121.5 15 1 1 1 1 125.8 16 0 0 0 − 2 137.9 17 − 1 − 1 1 − 1 136.2 18 1 1 − 1 1 139.7 19 1 1 1 − 1 146.1 20 1 − 1 − 1 1 145.1 21 0 0 0 0 166.3 22 0 0 2 0 132.8 23 1 − 1 − 1 − 1 112.4 24 0 0 0 2 142.4 25 0 0 0 0 165.5 26 1 − 1 1 − 1 121.3 27 1 − 1 1 1 132.3 28 1 1 − 1 − 1 141.4 29 0 0 − 2 0 127.1 30 − 1 − 1 − 1 1 146.8 ANOVA analysis According to the obtained statistical data and the ANOVA table, it is a good model that has the following two conditions: p-value < 0/05 In the selected model, R 2 should be closer to one (Table 3 ). Table 3 Quality of fitted to experimental data. R 2 0.9980 Adjusted R 2 0.961 Predicted R 2 0.9895 Adeq precision 71.9315 R 2 checks the quality of the experimental data with the model and the best value is one. Adj- R 2 is the modified value of R 2 , which also takes into account the degree of freedom (number of factors). The Predicted R 2 of 0.9897 agrees with the adjusted R 2 of 0.9961; That is, the difference is less than 0.2. Adeq Precision measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 71.932 indicates an adequate signal. This model can be used to navigate the design space. Statistical data were analyzed using the response level method and regression equation: Regression equation in encrypted units: \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{gathered} {\\text{CA }} = { 165}.{683 } + { 2}.{942}0{8}*{\\text{A }} + \\, - {1}.{41625}*{\\text{B }} + \\, 0.{994583}*{\\text{C }} + { 1}.{557}0{8}*{\\text{D }} + { 6}.{84688}*{\\text{AB }} + \\, - {2}.{44}0{63}*{\\text{AC }} + \\, 0.{938125}*{\\text{AD }} \\hfill \\\\ + - 0.{533125}*{\\text{BC }} + \\, - {8}.{14938}*{\\text{BD }} + \\, - {4}.{97438}*{\\text{CD }} + \\, - { 1}0.{1651}*{\\text{A}}^{{2}} + \\, - {1}0.{4151}*{\\text{B}}^{{2}} + \\, - {8}.{83635}*{\\text{C}}^{{2}} + \\, - {6}.{29135 }*{\\text{ D}}^{{2}} \\hfill \\\\ \\end{gathered}$$\\end{document} CA = 165.683 + 2.94208 ∗ A + - 1.41625 ∗ B + 0.994583 ∗ C + 1.55708 ∗ D + 6.84688 ∗ AB + - 2.44063 ∗ AC + 0.938125 ∗ AD + - 0.533125 ∗ BC + - 8.14938 ∗ BD + - 4.97438 ∗ CD + - 10.1651 ∗ A 2 + - 10.4151 ∗ B 2 + - 8.83635 ∗ C 2 + - 6.29135 ∗ D 2 The quadratic response level model is used to evaluate the effectiveness of the parameters and the accuracy of the model. The model F value of 528.44 indicates that the model is acceptable. There is only a 0.01% chance that an F value of this magnitude will occur due to the disturbance. P-values less than 0.0500 indicate that the model parameters are significant. In this case, A, B, C, D, AB, AC, AD, BD, CD, A 2 , B 2 , C 2 , and D 2 are acceptable parameters. Values that have p values greater than 0.0500, such as the BC parameter, indicate that this parameter does not affect the test conditions. Changing the model may improve your model if many model parameters are not affected. The Lack of Fit F-value of 3.16 indicates that a mismatch to a pure error is not acceptable. There is an 10.78% chance that a mismatch of the F-value of this magnitude will occur due to a disturbance. Non-significant disproportion is good because we want the model to fit Table 4 . Table 4 Analysis of variance. Source Sum of squares df Mean square F-value p-value Model 9135.54 14 652.54 528.44 < 0.0001 Significant A-H 2 O 207.68 1 207.68 168.19 < 0.0001 Significant B-EtOH 48.17 1 48.17 39.01 < 0.0001 Significant C-NH 3 24.40 1 24.40 19.76 0.0005 Significant D-PDMS 58.28 1 58.28 47.20 < 0.0001 Significant AB 748.02 1 748.02 605.77 < 0.0001 Significant AC 95.06 1 95.06 76.98 < 0.0001 Significant AD 14.06 1 14.06 11.39 0.0042 Significant BC 4.41 1 4.41 3.57 0.0783 Not significant BD 1062.76 1 1062.76 860.65 < 0.0001 Significant CD 396.01 1 396.01 320.70 < 0.0001 Significant A 2 2842.02 1 2842.02 2301.54 < 0.0001 Significant B 2 2983.34 1 2983.34 2415.98 < 0.0001 Significant C 2 2144.23 1 2144.23 1736.45 < 0.0001 Significant D 2 1085.76 1 1085.76 879.28 < 0.0001 Significant Residual 18.52 15 1.23 Lack of fit 15.99 10 1.60 3.16 0.1078 Not significant Pure error 2.53 5 0.5057 Cor total 9154.07 29 Diagnostics diagrams To troubleshoot the results obtained from the software the four graphs of normal probability, residuals vs. predicted, predicted vs. actual and Box-Cox plot for power transforms are used. In Fig. 2 a, the normal probability diagram shows that the residuals follow a normal distribution, therefore they follow a straight line. Even with normal data, expect some scatter. Figure 2 b (residuals vs. predicted) indicates that the residuals are bullish against the predicted response values. This plot tests the assumption of constant variance. The graph should have a random scatter and according to the graph the data follows a random scatter. Figure 2 c shows predicted vs actual. A graph of the predicted response values versus the actual response values. The purpose is to detect a value, or group of values, that are not easily predicted by the model. The Box-Cox chart is used to determine the strength of metamorphism consistent with experimental data (Fig. 2 d). The blue line in the diagram shows the model change and the green line shows the best lambda value. The red line indicates the 95% confidence interval associated with the best amount of lambda. It is said that a model is qualified for the blue conversion line between the red lines and the green line on the conversion curve to form a black and white curve. The graph shows that the blue transition line between the green line and the red line shows that the model matches the experimental results. Figure 2 Diagnostics diagrams ( a ) normal probability, ( b ) residuals vs. predicted, ( c ) predicted vs. actual and ( d ) Box-Cox plot for power transforms. Influence of single variables on contact angle Figure 3 shows the effect of the selected parameters on the contact angle. The graph follows a certain pattern for all parameters. With increasing water volume from 8 to 10, the contact angle gradually increased from 152.34° to 165.83°. Subsequently, with an increase from 10 to 12, the contact angle decreased from 165.83° to 158.45°. For the parameters of ethanol, ammonium hydroxide, and PDMS, the same changes occurred, i.e. the contact angle increased from − 1 to zero and decreased from zero to + 1. Figure 3 Influence of single variables on contact angle. The effect of binary variables on the contact angle In Fig. 4 , the interaction and three-dimensional diagrams of the model are obtained to estimate the interaction between the variables and the contact angle, while the other variables are kept at their zero levels and the others change in the experimental range. In Fig. 4 , as can be seen, the interaction of the binary parameters with each other is like the effect of the parameters individually. The difference between the interaction of the binary parameters with each other is in the angle between the two diagrams. The higher the angle of the two graphs, the greater the interaction, and if they are parallel, they have less or no interaction. According to the ANOVA table, the BC parameter is unacceptable, and the angle between the parameter BC is very small and they are almost parallel. Figure 4 The effect of binary variables on the contact angle. Process design optimization One of the purposes of test design is to optimize process parameters to obtain the highest contact angle. The size of the contact angle was determined to be 162°. According to the CCD design, the optimal conditions for the preparation of SiO 2 sol are shown in Fig. 5 . The experimental contact angle size for the optimized sample is compared with the predicted contact angle in Fig. 5 . The results showed that the size of the experimental contact angle corresponds well with the predicted contact angle and shows that the CCD surface response surface method is an efficient method for preparing ultra-waterproof coatings with a contact angle above 160°. Figure 5 Optimal values of process parameters. Identify the optimized sample Optimized sample according to statistical data analysis, as mentioned earlier, was prepared by StÖber method with TEOS as precursor, deionized water of hydrolyzing agent, solvent ethanol, ammonium hydroxide catalyst, and PDMS as a hydrophobic agent. As a result, the effect of experimental parameters on superhydrophobic coatings was investigated. Optimized sample contact angle The contact angle is the angle between the surface on which the liquid is located or the point of connection of the liquid on the surface. Static and dynamic contact angles are of its types. The method of measuring the contact angle is called the baseless droplet method. Can be used. The contact angle (CA) of the sliding angle (SA) obtained for the optimized sample is 162° and 5°, respectively (Fig. 6 ). Figure 6 Contact angle between the drop and the glass surface. XRD studies Figure 7 shows the X-ray diffraction pattern of nanoparticles prepared by the StÖber method. As shown in the figure, no diffraction peaks are observed except for broadband with a 24-degree center (JCPDS No. 0085-29) which represents a completely amorphous structure. But using Highscore plus software, it is shown that a small part of the sample has a crystalline structure. The marked peaks are related to hexagonal (JCPDS number 2147-080-01) and quadrilateral (JCPDS number 0430-079-01) and (JCPDS number 0513-082-01) crystal structures. Figure 7 XRD pattern of the optimized sample. FTIR spectrum Infrared spectroscopy (FT-IR) was performed at room temperature to investigate the chemical bonds created in the optimized sample. As shown in Fig. 8 , the peaks of 3440 cm −1 and 1624 cm −1 are symmetrical tensile vibration and flexural vibration of the O–H bond, respectively, due to the incomplete density of the silanol group 35 , 36 . The range of 400–1350 cm −1 , known as the fingerprint area, indicates silicon bonds. The peak 1095 cm −1 and 808 cm −1 represent the symmetric and asymmetric tensile vibrations of the Si–O–Si bond and the peak 466 cm −1 represents the flexural vibrations of the Si–O–Si 37 , 38 . The peak of 947 cm −1 is due to the flexural vibrations of the Si–OH bond 39 . As can be seen, the aging period causes the formation of a Si–O–Si tensile and bending bond in the sample. This creates a resistant coating on the surface of the glass. Figure 8 FT-IR spectrum of the optimized sample. SEM images Scanning electron microscope images of the optimized sample at different magnifications are shown in Fig. 9 . As seen in Fig. 9 a, the nanostructures have a spherical morphology with a size of about 250 nm. The reason for the growth of nanoparticles is due to the use of the hydrothermal method to create a suitable uneven surface on the glass surface. The shape and size of the nanostructure have an essential effect on creating a super hydrophobic coating. As can be seen, the spherical nanostructures have made a rough surface on the glass. Figure 9 b,c show the scanning electron microscope image of the surface and cross-section. The roughness of the glass surface indicates a rough surface for the super-hydrophobic coating. The growth process of this superhydrophobic coating is island-layer, which is a state between layer-by-layer growth and island growth, one or more monolayers are formed and then the islands are completed. Another name for this growth process is Stransky-Kristanov. In this growth mode, a mismatched network may be formed between the coated layer and the substrate. The grain size of the thin layer that is formed on the substrate depends on the speed and temperature of the layer. The thickness of this superhydrophobic coating is reported to be 1.06 µm. Figure 9 ( a ) SEM images of the optimized sample, ( b ) SEM images of the glass surface, and ( c ) cross-sectional SEM images of silica-coated glass. EDS studies Figure 10 a shows the X-ray energy diffraction spectrum for the optimal sample. As shown in the figure, the purely synthesized optimized sample is composed of silicon and oxygen elements. Figure 10 b shows the X-ray diffraction spectrum from the glass surface. Due to the use of PDMS as a hydrophobic agent on the glass surface, in addition to silicon and oxygen, carbon is also seen. The gold peak seen in the figure is due to the conductivity of the surface for SEM analysis. Figure 10 EDS for the ( a ) optimized sample and ( b ) glass surface. AFM images Atomic force microscopy images of the mean square root roughness for the optimized sample are estimated in Fig. 11 . As can be seen in the figure, rough surface roughness is created on the glass surface to create a super-hydrophobic coating. The maximum and minimum of these surface roughnesses were measured at 2.6 and 1.2 μm, respectively. The root means square roughness for the sample optimized by Gwyddion software was calculated to be 0.121 μm. Figure 11 AFM images of the optimized sample. DLS analysis As can be seen in the SEM images, the optimized sample has the same particle size distribution and DLS analysis was performed to determine the particle size distribution range. The particle size range is between 255 and 396.1 nm and, as shown in Fig. 12 , the particle size distribution diagram is very narrow. The average particle size is 291.456 nm. Coating thickness and surface roughness depend not only on particle size but also on size distribution. Therefore, as much as the particle size is properly distributed, the surface roughness increases and becomes uniform. Figure 12 Size distribution of the optimized sample. Chemical resistance test The stability of superhydrophobic coating was investigated in three media: acidic, neutral, and alkaline. 1 M KOH and 1 M HCl solutions were prepared for the resistance of the super-hydrophobic coating in alkaline and acidic environments, respectively. Figure 13 shows the effect of different environments on the contact angle. The superhydrophobic coating was immersed in 10 ml for 24 h. It was then dried at room temperature. As can be seen in the figure, the play environment (pH: 13.5) has a great impact on the coating so that the drop contact angle of the super-hydrophobic range reaches the boundary between hydrophobic and hydrophilic, in which case the result can be the game environment causes corrosion of the coating and the use of this coating in play environments is not recommended. The contact angle obtained from the acidic environment (pH: 1) indicates that the coating has good resistance and this feature can be very effective in coating against acid rain. The use of deionized water is used to accurately assess the strength of the coating when in contact with water. Therefore, to prevent the effect of temperature on the coating, this test was performed at a temperature of 25°. The contact angle resulting from the immersion of the coating in deionized water indicates the very good resistance of the coating in this environment. Figure 13 Effect of pH on the contact angle of the optimized sample."
} | 5,937 |
29599459 | null | s2 | 6,154 | {
"abstract": "Microorganisms engage in complex interactions with other organisms and their environment. Recent studies have shown that these interactions are not limited to the exchange of electron donors. Most microorganisms are auxotrophs, thus relying on external nutrients for growth, including the exchange of amino acids and vitamins. Currently, we lack a deeper understanding of auxotrophies in microorganisms and how nutrient requirements differ between different strains and different environments. In this Opinion article, we describe how the study of auxotrophies and nutrient requirements among members of complex communities will enable new insights into community composition and assembly. Understanding this complex network over space and time is crucial for developing strategies to interrogate and shape microbial communities."
} | 207 |
24885728 | PMC4032171 | pmc | 6,155 | {
"abstract": "Background The substrate, serving as carbon and energy source, is one of the major factors affecting the performance of microbial fuel cells (MFCs). We utilized BIOLOG system to rapidly screen substrates for electricigens, and further evaluated influence of these substrates on electricity generation of Shewanella loihica PV-4 in MFCs. Results Three of most favorable substrates (lactate acid, formic acid and cyclodextrin) with OD 590/750 of 0.952, 0.880 and 0.849 as well as three of most unfavorable substrates (galactose, arabinose and glucose) with OD 590/750 of 0.248, 0.137 and 0.119 were selected by BIOLOG system under aerobic conditions. The chronoamperometry results showed that MFCs fed with these substrates exhibited different current behaviors. Cyclic voltammograms results showed that arabinose, galactose and glucose promoted electron transfer from outer membrane c -Cyts of cells to the electrode surface. Lactic acid, formic acid and cyclodextrin produced lower quantity of electric charge of 10.13 C, 9.83 C and 10.10 C, the corresponding OD 600 value was 0.180, 0.286 and 0.152 in BES; while galactose, arabinose and glucose generated higher quantity of electric charge of 12.34 C, 13.42 C and 17.45 C, and increased OD 600 values were 0.338, 0.558 and 0.409 in BES. SEMs results showed that plenty of plump and stretched cells as well as appendages were observed when lactic acid, formic acid, and cyclodextrin were utilized as substrates, while sparse cells in short shape were obtained when galactose, arabinose and glucose were used as substrates. Conclusions These results suggest that substrate not only has important role in electrochemical performances of MFCs but also in biological properties of electricigens. Lactic acid, formic acid, and cyclodextrin beneficial for cell growth under aerobic conditions are unfavourable for planktonic cell growth and current generation under anaerobic conditions, while consumptions of galactose, arabinose and glucose adverse to cell growth under aerobic conditions are favourable for planktonic cell growth and current generation under anaerobic conditions due to the increase of cell numbers with more outer membrane c -Cyts transferring electrons between the electrode surface and cells.",
"conclusion": "Conclusions Lactic acid, formic acid, cyclodextrin, galactose, arabinose and glucose serving as electron donors for S. loihica PV-4 in MFCs were selected by BIOLOG system. Lactic acid, formic acid and cyclodextrin beneficial for cell growth under aerobic conditions were unfavourable for planktonic cell growth under anaerobic conditions and produced lower quantity of electric charge, while galactose, arabinose and glucose adverse to cell growth were favourable for planktonic cell growth under anaerobic conditions and generated higher quantity of electric charge. The electron donor played an important role not only in electrochemical performances of cells but also in cellular morphologies, especially the formation of appendages. Further researches including underlying formation mechanism and electrical properties of appendages are still necessary.",
"discussion": "Results and discussion Substrate screening results The substrate metabolism process of S. loihica PV-4 was indicated by a tetrazolium redox dye in GN2 MicroPlate. The specific pattern of color change on the plate provided an identifiable metabolic fringerprint. As shown in Figure 1 , some wells exhibited noticeable purple color in comparison with the control well (A1) with water as substrate after 25 h of aerobic culture. Most of negative wells showed no obvious color, it was indicated that the substrates in them are favorable for cellular growth and respiration. In addition, some wells exhibited half cyan color, it was suggested that the substrates in them are unfavorable. Combined with these OD 590/750 results, we chose three of the most favorable and unfavorable substrates to study substrate influence on electricity generation of S. loihica PV-4 in MFCs. They were D, L-lactic acid (E6, OD 590/750 = 0.952), formic acid (D4, OD 590/750 = 0.880), α-cyclodextrin (A2, OD 590/750 = 0.849), D-galactose (B4, OD 590/750 = 0.248), L-arabinose (A10, OD 590/750 = 0.137) and α-D-glucose (B6, OD 590/750 = 0.119). Figure 1 Profiling of S. loihica PV-4 cultures in GN2 MicroPlate using MicroLog software after 25 h of culture. Purple color: positive, no color: negative, plus and minus: mismatches. Chronoamperometry The electricity generation results of S. loihica PV-4 in bioelectrochemical system (BES) at a poised potential of 0.2 V for 25 h with lactic acid, formic acid, cyclodextrin, galactose, arabinose and glucose as substrates were shown in Figure 2 . With the addition of cells, an oxidative current was generated on ITO electrode, whereas no redox response was observed on the electrode without the addition of cells (data not shown). The oxidative current is ascribed to the electrical connection from outer membrane c -Cyts of cells to the electrode [ 21 - 24 ]. As shown in Figure 2 , the BES fed with different substrates in the presence of cells exhibited different current behaviors. When formate acid and cyclodextrin were used as substrates, an oxidative current of 0.19 μA cm −2 and 0.46 μA cm −2 was generated, and gradually grew with a broad current peak of 0.71 μA cm −2 and 0.57 μA cm −2 until ~11.4 h and ~3.2 h, then decreased slowly to a final current of 0.21 μA cm −2 and 0.23 μA cm −2 respectively. When lactic acid, galactose, arabinose and glucose were used as substrates, an immediate current peak of 1.76 μA cm −2 , 1.49 μA cm −2 , 1.58 μA cm −2 and 1.69 μA cm −2 was generated and gradually decreased or increased to a final current of 0.30 μA cm −2 , 0.56 μA cm −2 , 0.50 μA cm −2 and 0.79 μA cm −2 respectively. Similar chronoamperometry results were observed in the single-chamber, three-electrode electrochemical system with ITO as working electrode and S. loihica PV-4 as electricigens [ 22 , 25 ]. However, current was greatly enhanced on graphite (5 μA cm −2 ) [ 25 ], nanograss array boron-doped diamond (1.2 μA cm −2 ) [ 13 ], and ITO coated with polyaniline nanowire network electrodes (45 μA cm −2 ) [ 26 ] due to high surface roughness and nanostructured surface. It was reported that an important aspect of achieving high power density was having a low internal resistance [ 27 ]. The total internal resistance consists of anodic, membrane, cathodic, and electrolyte resistance [ 28 ]. Among these, anodic resistance is the main limiting factor in internal resistance in this single-chamber, three-electrode electrochemical system where the cathode is platinum wire. The low current generated on ITO electrode was ascribed to increase of anodic resistance from small population of bacteria attached to the plane electrode surface [ 27 ]. However, ITO is widely used for spectroelectrochemical characterization of purified redox proteins and whole microbial cells due to its excellent optoelectronics properties [ 22 , 25 ]. Herein, it was chosen for the study of substrate effect on biological and electrochemical properties of S. loihica PV-4. Figure 2 Substrate effect on the electricity generation of \n S. loihica \n PV-4 in MFCs. Cylic voltammetry The effect of substrates on electron transfer between S. loihica PV-4 cells and ITO electrode was evaluated by cyclic voltammograms (CVs), after polarizing the electrode for 25 h. As shown in Figure 3 , there were no obvious redox waves on ITO electrode in the absence of substrate. After the addition of substrate, pairs of well-defined redox waves were observed on the electrode. The current generation was ascribed to the oxidation of organic compounds coupled to reduction of electron acceptors by cells. For formic acid, cyclodextrin and lactic acid, CVs showed sharp reductive peaks at −0.298 V, −0.318 V, −0.332 V, and broad oxidative peaks at −0.060 V, −0.082 V, −0.094 V. For arabinose, galactose and glucose, quasi-reversible CVs with reductive peaks at −0.336 V, −0.338 V, −0.378 V and oxidative peaks at −0.124 V, −0.168 V, −0.124 V were exhibited on the electrode. These CVs results confirm an outer membrane c -Cyts-mediated electron transfer to the electrode surface [ 13 , 22 ]. The cells inoculated in the presence of arabinose, galactose and glucose exhibited higher redox peak currents as compared to those using formic acid, cyclodextrin and lactic acid as substrates. It was indicated that arabinose, galactose and glucose promoted electron transfer from outer membrane c -Cyts of cells to the electrode surface and cells fed with these substrates had superior electrochemical performances. These CVs results were in accordance with the chronoamperometry results. Figure 3 CVs of S. loihica PV- 4 obtained on ITO electrode in the absence (blank control) and presence of substrates. Scan rate: 0.01 mV s −1 . Quantity of electric charge and cell growth To further explore the substrate effect on electricity generation, the total quantity of electric charge ( Q ) was calculated by integrating each I-T curve with respect to time. The formula was Q = ∫ 0 t Idt [ 29 , 30 ]. The interrelationship between total electric charge and planktonic cell growth under anaerobic conditions with lactic acid, formic acid, cyclodextrin, galactose, arabinose and glucose as substrates in BES after 25 h was shown in Figure 4 . When lactic acid, formic acid and cyclodextrin were used as substrates, the quantity of electric charge of MFC in 25 h was 10.13 C, 9.83 C and 10.10 C respectively; the corresponding OD 600 value representing planktonic cell growth was 0.180, 0.286 and 0.152. In comparison, when galactose, arabinose and glucose were used as substrates, increased quantity of electric charge of 12.34 C, 13.42 C and 17.45 C, and increased OD 600 values of 0.338, 0.558 and 0.409 were observed. It was suggested that lactic acid, formic acid and cyclodextrin beneficial for cell growth of S. loihica PV-4 under aerobic conditions were unfavorable for electricity generation and planktonic cell growth under anaerobic conditions, while galactose, arabinose and glucose adverse for cell growth under aerobic conditions were favorable for electricity generation and planktonic cell growth under anaerobic conditions. These results were in accordance with the above CVs results that BES fed with galactose, arabinose and glucose had superior electrochemical performances. This was ascribed to the increase of cell numbers with more outer membrane c -Cyts transferring electrons between the electrode surface and cells. Figure 4 Substrate effect on quality of electric charge and cell growth of \n S. loihica \n PV-4 in MFCs after 25 h of culture."
} | 2,685 |
34671322 | PMC8521193 | pmc | 6,158 | {
"abstract": "Identifying the enzymes involved in lignin degradation by bacteria is important in studying lignin valorization to produce renewable chemical products. In this paper, the catalytic oxidation of lignin by a novel multi-copper polyphenol oxidoreductase (OhLac) from the lignin degrader Ochrobactrum sp. J10 was explored. Following its expression, reconstitution, and purification, a recombinant enzyme OhLac was obtained. The OhLac enzyme was characterized kinetically against a range of substrates, including ABTS, guaiacol, and 2,6-DMP. Moreover, the effects of pH, temperature, and Cu 2+ on OhLac activity and stability were determined. Gas chromatography-mass spectrometer (GC-MS) results indicated that the β-aryl ether lignin model compound guaiacylglycerol-β-guaiacyl ether (GGE) was oxidized by OhLac to generate guaiacol and vanillic acid. Molecular docking analysis of GGE and OhLac was then used to examine the significant amino residues and hydrogen bonding sites in the substrate–enzyme interaction. Altogether, we were able to investigate the mechanisms involved in lignin degradation. The breakdown of the lignocellulose materials wheat straw, corn stalk, and switchgrass by the recombinant OhLac was observed over 3 days, and the degradation results revealed that OhLac plays a key role in lignin degradation.",
"conclusion": "Conclusion In this paper, a novel bacterial multicopper oxidase, OhLac, from Ochrobactrum sp. J10 was identified and characterized. After heterologous expression and purification, we noted that OhLac differed from other multicopper oxidases reported previously. In particular, it had a lower molecular weight and two structural domains. Notably, the results proved that this small multicopper oxidase has the potential to be useful in the oxidation and degradation of lignin, playing a key role in the process. Moreover, the cleavage of the β-O-4 linkage in lignin was explored and discussed as part of the oxidative process. Improved knowledge of bacterial enzymes and their roles in lignin degradation will have a significant impact on a wide array of biotechnologies focused on lignin degradation.",
"introduction": "Introduction The natural constituents of plant biomass are cellulose, lignin, hemicellulose, pectin, and so on. Among these, the aromatic heteropolymer lignin, which forms a natural physical barrier to the hydrolysis of cellulose, poses a great challenge in the utilization of lignocellulosic biomass ( Yang and Lü, 2021 ). Many commercial chemical and physical methods aimed at lignin degradation are non-specific, energy consuming, and harmful to the environment. Biological methods involving microbial enzymes to oxidize and break down lignin structures have been studied extensively. Ligninolytic enzymes such as lignin peroxidases, manganese-dependent peroxidases, laccases, and dye-decolorizing peroxidases are effective in lignin degradation ( Zhu et al., 2020 ; Kuppuraj et al., 2021 ). The multi-copper oxidase group forming part of a large class of enzymes ( Janusz et al., 2017 ), especially laccases (benzenediol: oxygen oxidoreducutases; EC 1.10.3.2), are known as one of the important enzymes of wood-destroying microorganisms. Notably, these enzymes can oxidize substrates by four redox-active copper ions as the cofactors and produce only water as a by-product; therefore, they are termed “green enzymes” ( Riva, 2006 ). Laccases from fungi, bacteria, plants, and insects vary greatly in structure, molecular weight, or oligomeric state ( Mayer and Staples, 2002 ; Navaneetha et al., 2011 ; Mate and Alcalde, 2015 ). Most characterization of fungi laccases has been conducted in Trametes versicolor and Pycnoporus cinnabarinus , where they have been shown to play important roles in lignin degradation ( Youn et al., 1995 ; Eggert et al., 1997 ). Moreover, laccases have been shown to be abundant in bacteria, such as Bacillus pumilus , Bacillus licheniformis , and Escherichia coli , in which they are active against a wide range of substrates ( Hullo et al., 2001 ; Xu et al., 2007 ; Chang et al., 2014 ). Traditional laccases were previously reported to consist of three structural domains and contained four copper ions in the active site: T1 Cu was responsible for the characteristic blue color and caused oxidation of substrate molecules by one-electron abstraction ( Jones and Solomon, 2015 ); T2 Cu and a pair of T3 Cu formed a trinuclear cluster in which molecular oxygen was reduced to water ( Mate and Alcalde, 2015 ). Recently, laccases from bacteria that lack one of the three structural domains were identified and designated as “small laccases” because of their sequence similarity but smaller size compared with traditional laccases ( Machczynski et al., 2004 ; Majumdar et al., 2014 ). Small laccases, such as SLAC from Streptomyces coelicolor and Ssl1 from Streptomyces sviceus , were extensively characterized and reported to be effective lignin-degrading enzymes ( Gunne et al., 2014 ; Majumdar et al., 2014 ). Furthermore, these enzymes were demonstrated to have high oxidizing ability, thermal stability, and pH versatility. A novel polyphenol oxidase, P-PPO, with a lower molecular weight (33.4 kDa) was found and amplified from Paenibacillus sp., but unfortunately no activity was found with all the substrates tested against this enzyme ( Granjatravez et al., 2018 ). Therefore, polyphenol oxidases from different kinds of bacteria require further investigation. In addition, detailed information about the lignin-degrading mechanism of these enzymes has rarely been studied using lignin model compounds and raw lignocellulosic biomass as substrates. In the present study, a novel multi-copper polyphenol oxidoreductase, OhLac, was amplified and identified from the lignin-degrading bacteria Ochrobactrum sp. J10, which was isolated from rotten wood in Qinling, China, and reported in our previous study ( Yang et al., 2017 ). The enzyme activity and biochemical characterization of OhLac were studied using a broad range of substrates. Furthermore, the lignin cleavage mechanism of OhLac was explored and assessed in the context of its lignin-degrading abilities.",
"discussion": "Discussion The polyphenol oxidoreductase OhLac from Ochrobactrum sp. J10 was demonstrated to be a potential lignin-degrading enzyme. The results of the kinetic characterization indicated that OhLac might show better activity and potential for certain lignin-degrading applications. It was well known that the traditional fungal and bacterial laccases contained three domains and four copper atoms in their active center, whose molecular weights were about 60 kDa, such as CotA ( Koschorreck et al., 2008 ). In addition, another form of the polyphenol oxidoreductase was found with two domains (about 30 kDa) in bacteria or called “small laccases” ( Majumdar et al., 2014 ). The property of small laccases was reported to be markedly different from that of traditional laccases. It had been proved that they had activities against a wide range of phenolic compounds ( Prins et al., 2015 ). Besides, they had the advantages of high oxidizing power, thermal stability, and pH versatility ( Lee et al., 2019 ). However, more detailed biochemical and structural studies of various smaller size laccases should be conducted and explored deeply to promote their application to lignin degradation. Guaiacylglycerol-β-guaiacyl ether is a typical phenolic model compound containing the β-O-4 bond. It has been reported that the intersubunit content of β-O-4 bonds is more than 50% in lignin ( Villaverde et al., 2009 ). These peaks (guaiacol and vanillic acid) appeared to correspond to the Cα-Cβ oxidative products of GGE when degraded by OhLac. Similarly, OcCueO has been reported to decompose lignosulfonate to form vanillin acid as a product, which indicates cleavage of the Cα-Cβ bond in lignin ( Granjatravez et al., 2018 ). The small laccases from Streptomyces (SCLAC) were studied to investigate the degradation of the phenolic β-O-4 lignin model compound (LM-OH), and vanillin was identified as a degradation product because of Cα-Cβ bond cleavage. SCLAC could oxidize the non-phenolic β-O-4 lignin model compound (LM-OMe) to form the corresponding ketone product ( Majumdar et al., 2014 ). We found that there was a hydrogen bond between the substrate GGE and the amino acid THR134 of OhLac. Specifically, the THR134 residue could form a hydrogen bond with the hydroxyl group attached to the Cα position in GGE, which might offer atoms as hydrogen bond donors and acceptors ( Yang et al., 2015 ). The results indicated that these amino residues were very important putative catalytic sites. The contribution of every active site to enzyme activity will be explored in future work. The above results from the studies of degradation and docking of lignin model compounds indicated that OhLac was available to oxidize lignin. Therefore, lignin cleavage by some bacteria might be related to the action of multicopper oxidases. Laccase may only be responsible for the radical initiation step, while the downstream Cα-Cβ bond cleavage may be the result of spontaneous reaction ( Leonowicz et al., 2001 ; Majumdar et al., 2014 ). The oxidation of the lignin model compound by a fungal laccase led to the formation of the corresponding ketone product instead of Cα-Cβ bond cleavage ( Li et al., 1999 ). Laccases catalyze oxidation of lignin, initially producing a phenoxy radical ( Perna et al., 2019 ). It had often been reported that Cα-oxidation, rather than ether bond cleavage, was the main result of laccase-catalyzed lignin degradation system ( Heap et al., 2014 ; Hilgers et al., 2019 ). A non-phenolic lignin model, veratrylglycerol-β-guaiacyl ether (VBG), was used to explore the degradation of lignin by a laccase/HBT system. The results showed that the Cα-ketone analog of VBG (VBGox) and two ether cleavage products were identified, which indicated Cα-oxidation cleavage ( Hilgers et al., 2020 ). In this study, a mixture of oxidative degradation products was formed, which were not fully characterized. However, the products were identified and characterized as guaiacol and vanillic acid. Therefore, it was supposed that during the oxidation of lignin by OhLac, oxidation of the α-carbon center leads to the formation of the ketone product, and then continued oxidation causes the cleavage of the Cα-Cβ bond of lignin ( Figure 8 ). OhLac is thought to breakdown dimeric lignin GGE by cleaving the Cα-Cβ bond and to produce guaiacol (L2) and vanillic acid (L3). FIGURE 8 The presumed cleavage process of lignin model compound GGE degraded by OhLac."
} | 2,652 |
27355054 | PMC4897565 | pmc | 6,159 | {
"abstract": "Microbial metal reduction can be a strategy for remediation of metal contaminations and wastes. Bacteria are capable of mobilization and immobilization of metals and in some cases, the bacteria which can reduce metal ions show the ability to precipitate metals at nanometer scale. Biosynthesis of nanoparticles (NPs) using bacteria has emerged as rapidly developing research area in green nanotechnology across the globe with various biological entities being employed in synthesis of NPs constantly forming an impute alternative for conventional chemical and physical methods. Optimization of the processes can result in synthesis of NPs with desired morphologies and controlled sizes, fast and clean. The aim of this review is, therefore, to make a reflection on the current state and future prospects and especially the possibilities and limitations of the above mentioned bio-based technique for industries.",
"conclusion": "5. Conclusion Bio-based approaches are still in the development stages, and stability and aggregation of the biosynthesized NPs, control of crystal growth, shape, size, and size distribution are the most important experienced problems. Furthermore, biologically synthesized NPs in comparison with chemically synthesized ones are more polydisperse. The properties of NPs can be controlled by optimization of important parameters which control the growth condition of organisms, cellular activities, and enzymatic processes (optimization of growth and reaction conditions). Mechanistic aspects have not been clearly and deeply described and discussed. Thus, more elaborated studies are needed to know the exact mechanisms of reaction and identify the enzymes and proteins which involve nanoparticle biosynthesis. The large-scale synthesis of NPs using bacteria is interesting because it does not need any hazardous, toxic, and expensive chemical materials for synthesis and stabilization processes. It seems that by optimizing the reaction conditions and selecting the best bacteria, these natural nanofactories can be used in the synthesis of stable NPs with well-defined sizes, morphologies, and compositions.",
"introduction": "1. Introduction Nanoscience and nanotechnology has attracted a great interest over the last few years due to its potential impact on many scientific areas such as energy, medicine, pharmaceutical industries, electronics, and space industries. This technology deals with small structures and small-sized materials of dimensions in the range of few nanometers to less than 100 nanometers. Nanoparticles (NPs) show unique and considerably changed chemical, physical, and biological properties compared to bulk of the same chemical composition, due to their high surface-to-volume ratio. NPs exhibit size and shape-dependent properties which are of interest for applications ranging from biosensing and catalysts to optics, antimicrobial activity, computer transistors, electrometers, chemical sensors, and wireless electronic logic and memory schemes. These particles also have many applications in different fields such as medical imaging, nanocomposites, filters, drug delivery, and hyperthermia of tumors [ 1 – 4 ]. An important area of research in nanoscience deals with the synthesis of nanometer-size particles of different morphologies, sizes, and monodispersity [ 5 ]. In this regard, there is a growing need to develop reliable, nontoxic, clean, ecofriendly, and green experimental protocols for the synthesis of NPs [ 6 – 12 ]. One of the options to achieve this objective is to use natural processes such as use of enzymes, microbial enzymes, vitamins, polysaccharides, biodegradable polymers, microorganisms, and biological systems for synthesis of NPs. One approach that shows immense potential is based on the biosynthesis of NPs using bacteria (a kind of bottom up approach) [ 6 , 7 , 11 ]. The objects of recent studies tend to provide a controlled and up-scalable process for biosynthesis of monodispersed and highly stableNPs. Thus, a wide number of bacterial species have been used in green nanotechnology to research alternative methods for the synthesis of NPs. Researchers have started to use biomass or cell extracts of bacteria for synthesizing NPs. Bacteria are considered as a potential biofactory for the synthesis of NPs like gold, silver, platinum, palladium, titanium, titanium dioxide, magnetite, cadmium sulphide, and so forth. Some well-known examples of bacteria synthesizing inorganic materials include magnetotactic bacteria and S layer bacteria. Most metal ions are toxic for bacteria, and, therefore, the bioreduction of ions or the formation of water insoluble complexes is a defense mechanism developed by the bacteria to overcome such toxicity [ 13 – 16 ]. In this review, most of the bacteria used in nanoparticle biosynthesis are shown. The aim of this paper is, therefore, to make a reflection on the current state and future prospects and especially the possibilities and limitations of the above mentioned bio-based technique for industries."
} | 1,250 |
40272017 | PMC12036490 | pmc | 6,160 | {
"abstract": "ABSTRACT Multispecies biofilms are communities composed of different microorganisms embedded in an auto-synthesized polymeric matrix. Pseudomonas aeruginosa and Burkholderia cenocepacia are two multidrug-resistant and biofilm-forming opportunistic pathogens often found in the lungs of people living with cystic fibrosis. In this context, planktonic, static, and dynamic biofilms and in vivo models of both species were optimized in this work to understand their population dynamics, disposition, virulence, and antibiotic susceptibility. From the coculture models optimized in this work, we determined that B. cenocepacia grows in a clustered, aggregative manner at the bottom layers of biofilms, in close contact with P. aeruginosa , that tends to occupy the top layers. Their coexistence increases virulence-related gene expression in both species at early stages of coinfection and in in vivo models, while there was a general downregulation of virulence-related genes after longer coexistence periods as they eventually reach a non-competitive stage during chronic infections. When evaluating antimicrobial susceptibility, a decrease of antimicrobial tolerance was observed in both species when co-cultured. These findings shed light on the differential behavior of P. aeruginosa and B. cenocepacia in dual-species systems, stressing the relevance of multispecies studies in the clinical context.",
"introduction": "Introduction Bacterial biofilms are a particular growth strategy in which sessile bacterial cells are embedded in a self-produced polymeric matrix mainly composed of polysaccharides, proteins, and extracellular DNA (eDNA). When forming biofilms, bacteria show virulent and survival-promoting phenotypic traits in hostile environments, including drug resistance mechanisms, and they become extremely difficult to eradicate [ 1–3 ]. Multispecies biofilms are aggregates consisting of different microorganisms interwoven or in proximity, allowing potential inter-species and context-dependent interactions [ 4 ]. In the case of cystic fibrosis (CF), the lung airways of people living with this condition are infected by polymicrobial communities that change throughout the patient’s lifetime [ 5 ]. In this study, we will focus on Pseudomonas aeruginosa and Burkholderia cenocepacia as it has been described that people with CF infected with P. aeruginosa are more prone to developing secondary infections with opportunistic pathogens such as B. cenocepacia [ 6 ]. P. aeruginosa is a Gram-negative opportunistic pathogen, which establishes in immunocompromised individuals such as those with CF. The key to its infective potential is its ability to form biofilms, and one of the most extensively studied scenarios illustrating the link between biofilms and chronic infections is the presence of P. aeruginosa in the lungs of people with CF, as 70% of these develop P. aeruginosa chronic respiratory infection [ 7 ]. B. cenocepacia is a ubiquitous Gram-negative bacterium that can occasionally be found infecting the lungs of people with CF, but it is associated with reduced patient survival and an accelerated loss of lung function. Moreover, approximately 20% of individuals with CF who are infected with B. cenocepacia develop fatal Cepacia syndrome, an acute lung failure that generally leads to necrotizing pneumonia [ 8 , 9 ]. P. aeruginosa forms mucoid biofilms with B. cenocepacia , which promote a strong network of interactions as well as the exchange of genetic material [ 10 , 11 ]. When considering the in vitro study of two different bacterial species, it is crucial to choose an adequate experimental set-up, inoculum ratio, incubation time, and culture media that allow the coexistence of both species [ 12 ], which often translates into the need for a previous coculture optimization. Literature is extensive when it comes to optimal growth of P. aeruginosa in multi-species cultures with other bacterial or fungal species [ 13–17 ]. Mixed cultures of P. aeruginosa and B. cenocepacia have already been studied, where their contact-independent interactions in planktonic conditions [ 18 , 19 ], contact-dependent interaction when forming colonies or static biofilms [ 20 , 21 ], altered toxin susceptibility in planktonic conditions [ 22 ] or murine host immune response to a mixed infection were evaluated [ 23 ]. However, the three-dimensional spatial distribution of P. aeruginosa and B. cenocepacia in mixed cultures, their altered tolerance to clinically used antibiotics when in coculture, and the expression of strain-specific virulence-related genes under different experimental conditions remain unexplored. In vivo models play a pivotal role in modern biomedical research, offering an essential platform for studying interspecies and host-pathogen interactions within the context of a whole organism. Galleria mellonella , or greater wax moth, is a popular animal model for the study of virulence and pathogenicity of different bacteria [ 24 , 25 ]. It is part of the Lepidopetra order and has a life cycle of approximately 8 weeks, with their larval stage being the one where bacterial infection is commonly tested. G. mellonella larvae are easily manipulated, are inexpensive to purchase and easily bred, present a low biohazard risk, and are not subjected to ethical regulations as they do not possess nociceptors [ 26 ]. In this study, we initially focused on achieving a stable and long-lasting coculture of P. aeruginosa PAO1 and B. cenocepacia by optimizing the growth conditions in planktonic, static, and dynamic biofilms. We evaluated the bacterial distribution under dynamic conditions and the biofilm formation process over time. Additionally, we examined the effects of two antibiotics on single- and dual-species biofilms to determine the impact of a coculture on antibiotic tolerance. Also, we investigated the impact of coinfection on the survival of G. mellonella larvae. Finally, we analyzed the virulence response of each species when grown in the different optimized set-ups to elucidate their contribution to biofilm composition and their potential effect on the health outcome of people with CF.",
"discussion": "Discussion The increasing emergence of antimicrobial-resistant bacteria is currently a critical worldwide threat. Biofilms play a crucial role in chronic bacterial infections, and the fact that most of these infections are polymicrobial challenges of the antimicrobial therapy treatment and thus lead to poorer clinical outcomes [ 6 , 53 ]. In this study, we have examined the effects of a P. aeruginosa and B. cenocepacia coculture in terms of viability, spatial distribution, antibiotic susceptibility, and virulence. The static biofilm set-up offers many methodological advantages compared to other in vitro biofilm culturing techniques. However, its static nature leads to an accumulation of metabolic waste products, bactericidal molecules, and a limited nutrient availability [ 29 , 40 ]. In this context, the B. cenocepacia viability differences observed between single- and dual-species biofilms ( Figure 1 ) are most likely due to the bactericidal molecules and enzymes secreted by P. aeruginosa at early stages of biofilm formation and in planktonic conditions (Figure S3), as also described by previous studies [ 19 , 23 ]. In addition, its rapid growth rate compared to that of B. cenocepacia (Figure S4) would allow it to dominate the coculture in static conditions [ 23 , 54 ]. Moreover, the increase on the viability of B. cenocepacia observed with the decrease of the initial inoculum of P. aeruginosa ( Figure 1(a) ) is also linked to a scarce concentration of bactericidal molecules at early stages of biofilm formation, when B. cenocepacia is more susceptible to them as the biofilm is not completely formed. The interspecific competition between P. aeruginosa and B. cenocepacia is mediated by antagonistic interactions including context-dependent T6SS-mediated delivery of bactericidal effectors [ 55 , 56 ]. P. aeruginosa outcompetes B. cenocepacia through the production of antimicrobial compounds such as pyocyanin, pyocins, and rhamnolipids, as well as superior iron acquisition via siderophores like pyoverdine and pyochelin [ 22 , 57 ]. Furthermore, P. aeruginosa interferes with B. cenocepacia quorum sensing, disrupts its metabolic processes [ 58 ], and exhibits competitive dominance in biofilm-associated polymicrobial infections such as those in people living with CF. When we monitored the virulence gene expression of P. aeruginosa and B. cenocepacia in static biofilm conditions ( Figure 7(c,d) ), we observed a general overexpression at early stages of biofilm formation. These genes encode bactericidal compounds like exotoxin A ( toxA ), exotoxin S (e xoS ), and hemolytic phospholycin C ( plcH ), potentially reducing the B. cenocepacia population at the beginning of the biofilm formation process. In B. cenocepacia , we observed a significant overexpression of the gspE gene, which encodes the GspE T2SS ATPase responsible for supplying the energy needed for virulence factor translocation. This suggests that B. cenocepacia is competing with P. aeruginosa in established static biofilms, although this competition does not result in a decrease in CFU/mL of the latter. Surprisingly, the studied P. aeruginosa genes were significantly downregulated when in dual-species mature static biofilms, meaning that the presence of B. cenocepacia reduces the virulence of P. aeruginosa in well-established biofilm cocultures, eventually reaching an equilibrium point where competition ceases. It is known that the media composition, oxygen availability, and the initial inoculum ratio influence the stability, bacterial fitness, and the composition of biofilms and planktonic cultures [ 39 , 59 , 60 ]. In this study, TSB was chosen as the growth medium due to its reproducibility, affordability, and ease of preparation, and experiments were performed in aerobic conditions. When different media supplements were tested in static biofilms ( Figure 1(b) ), the addition of 5% BSA enhanced the viability of B. cenocepacia in coculture with P. aeruginosa , and the same results were obtained in planktonic cultures ( Figure 2 ). This reflects that BSA plays a role on reducing the infective potential of P. aeruginosa , by potentially binding and sequestering key P. aeruginosa quorum-sensing molecules or other bactericidal molecules as seen in other studies [ 13 , 60 , 61 ]. The study of the P. aeruginosa − B. cenocepacia interaction in planktonic conditions revealed that they clearly establish a competitive relationship. Distribution-wise, it was interesting to observe that B. cenocepacia tends to grow in an aggregated manner in planktonic cultures ( Figure 2 ), while P. aeruginosa is dispersed throughout the coculture. Aggregation in B. cenocepacia [ 62–64 ], which indicates that the aggregation of B. cenocepacia observed in Figure 2 is indeed caused by a stressful competitive environment provoked by the presence of P. aeruginosa . In addition, when monitoring the B. cenocepacia gene expression pattern in planktonic conditions ( Figure 7(b) ), the cblA gene was significantly upregulated. CblA is the major subunit of the cable pili, and it has been linked to bacterial attachment to cells as well as to B. cenocepacia [ 65 ], which confirms the shift of B. cenocepacia into a defensive phenotype that implies self-aggregation. The l -Ornithine N 5 -Oxygenase is a crucial enzyme in the ornibactin biosynthesis pathway, and it is encoded by the pvdA gene, which is the most upregulated of the studied genes in planktonic conditions. The pvdA gene has been shown to be required for effective colonization and persistence in the lung [ 48 ], and it potentially contributes to inter-species competition [ 66 ]. From the P. aeruginosa point of view, most of the studied genes, which encode for bacteriocin molecules, were upregulated, which explains its dominance in the coculture. Flow cell dynamic systems for in vitro biofilm study offer an exceptional balance between mimicking the physiological conditions while being economic and adaptable [ 40 ]. Although it requires more complex settings than static biofilms, flow systems prevent the accumulation of toxins, metabolites, and waste products, allowing the formation of a stable dual-species biofilm. In this study, we have established a dual-species biofilm in a dynamic set-up that is stable over time, where both species maintain their viability for more than 96 h. When observing the spatial distribution of both strains in dynamic conditions, we have determined that B. cenocepacia forms single-species clusters at the bottom of the biofilm, and P. aeruginosa covers the spaces between and above those clusters, existing segregation between both species ( Figure 3 ) in a similar manner to the observed distribution in planktonic cocultures. In addition, B. cenocepacia attaches to the glass surface at early experimental stages and forms clusters that do not vary greatly over time, while P. aeruginosa colonizes the surface of these clusters and steadily grows all over them ( Figure 4 ). The fact that this disposition is contrary to depletion aggregation mechanisms may be explained by the fact that both species have T6SS, and thus, they grow separately as a means of protection [ 63 ]. Additionally, both species retain their shape and structure throughout the entire experimental timespan, suggesting no contact-dependent killing between them, although it should be further studied. However, the restricted growth of B. cenocepacia indicates the potential presence of contact-independent killing by P. aeruginosa , as proven in planktonic conditions. Interestingly, in dynamic flow systems, the presence of both P. aeruginosa and B. cenocepacia in a mature biofilm results in a neutral and even downregulated virulence-related gene expression ( Figure 7(e,f) ), which indicates that both species have reached a balanced dual-species biofilm and that the competitive nature of their interaction fades away over time, allowing their coexistence. G . mellonella has been used as an in vivo model for a wide variety of purposes, from toxicity testing of novel compounds to bacterial infection development. In accordance with other multispecies coinfection studies involving P. aeruginosa [ 67 ], we have determined that the presence of P. aeruginosa and B. cenocepacia as a coinfection significantly compromises larval survival compared to single infections ( Figure 6 ), thus increasing the infection severity. In addition, Bragonzi et al . [ 23 ] hypothesized that an increased virulent response was responsible for their observed rise in inflammatory response in mice models when coinfected with P. aeruginosa and B. cenocepacia [ 23 ]. In this study, we have indeed proven that there is a significant overexpression of virulence-related genes toxA and vgrG2b for P. aeruginosa when coinfecting G. mellonella , which also explains the significant larval survival decrease in coinfection compared to single-infected larvae ( Figure 7(g,h) ). The biofilm lifestyle has been linked to an increase in antimicrobial tolerance, as microbial growth rates decrease and the exopolysaccharide matrix acts as a physical barrier that protects them [ 68 ]. TOB and CPX belong to the aminoglycoside and fluoroquinolone antibiotic groups, respectively, and are commonly used to combat infections in people living with CF [ 69 ]. Previous studies have shown that the presence of multiple species on a bacterial biofilm may increase P. aeruginosa tolerance to antimicrobial therapies [ 13 ]. However, the opposite effect was observed in our work when it comes to the presence of B. cenocepacia in the dual-species biofilm ( Figure 5 ), as also reported in coculture with Streptococcus and Prevotella species [ 70 ]. We have found that P. aeruginosa and B. cenocepacia are more sensitive to the antibiotic effect when in dual-species biofilm compared to single-species biofilms. It is worth mentioning that the MIC of both bacterial species greatly differs, being that of B. cenocepacia much higher than that of P. aeruginosa . In addition, the spatial distribution of both bacteria in the biofilm ( Figure 3 ), where P. aeruginosa is on top and B. cenocepacia is at the bottom, may also influence the antibiotic susceptibility of the two strains [ 34 ]. Interestingly, what we have also found is an increase in the CFU/mL of B. cenocepacia obtained after TOB treatment at effective doses for P. aeruginosa . Subinhibitory concentrations, or concentrations below the MIC for a certain bacterium, have been shown to enhance the growth or biofilm formation capacity of different bacterial strains [ 71 , 72 ]. This finding highlights that treatments targeting P. aeruginosa in clinical settings may promote the growth of B. cenocepacia due to subinhibitory effects, as its MIC is higher than that of P. aeruginosa . Previous studies have shown that B. cenocepacia and P. aeruginosa exhibit differences in virulence factor production and toxin susceptibility when grown in CF-specific media like Synthetic Cystic Fibrosis Medium (SCFM) compared to standard nutrient-rich media [ 22 , 73 ]. These findings emphasize the importance of media selection, highlighting that the use of TSB in this study provides more general growth conditions than SCFM, which represents a limitation. Additionally, oxygen availability has been proven to alter virulence-related gene expression [ 74 ], and since CF infections occur in microaerophilic environments, future studies should explore the differential behavior of mixed cultures under such conditions. Finally, interspecies interactions are strain-dependent, and different clinical isolates may yield varying results, making this an important aspect to investigate in future research. In conclusion, we have demonstrated the potential of BSA to maintain a balanced dual-species in vitro coculture of P. aeruginosa and B. cenocepacia . Additionally, we have characterized the population dynamics of planktonic cocultures and optimized the growth conditions in static biofilms to determine the shifts in antibiotic tolerance when in coculture. Also, we investigated the spatial distribution of both species in dynamic flow BiofilmChip systems over time, determined that the coculture of both strains worsens the infection outcome in the G. mellonella in vivo model, and monitored bacterial virulence-related gene expression across all studied models. This study provides valuable insights into the establishment of P. aeruginosa and B. cenocepacia dual-species biofilms in vitro , as well as their virulence patterns during different growth phases and in vivo , which could aid in understanding phenotypes derived from this clinically challenging bacterial coinfection."
} | 4,765 |
36091416 | PMC9449976 | pmc | 6,161 | {
"abstract": "Aiming at the problems of slow convergence and easy fall into local optimal solution of the classic ant colony algorithm in path planning, an improved ant colony algorithm is proposed. Firstly, the Floyd algorithm is introduced to generate the guiding path, and increase the pheromone content on the guiding path. Through the difference in initial pheromone, the ant colony is guided to quickly find the target node. Secondly, the fallback strategy is applied to reduce the number of ants who die due to falling into the trap to increase the probability of ants finding the target node. Thirdly, the gravity concept in the artificial potential field method and the concept of distance from the optional node to the target node are introduced to improve the heuristic function to make up for the fallback strategy on the convergence speed of the algorithm. Fourthly, a multi-objective optimization function is proposed, which comprehensively considers the three indexes of path length, security, and energy consumption and combines the dynamic optimization idea to optimize the pheromone update method, to avoid the algorithm falling into the local optimal solution and improve the comprehensive quality of the path. Finally, according to the connectivity principle and quadratic B-spline curve optimization method, the path nodes are optimized to shorten the path length effectively.",
"conclusion": "Conclusion The ant colony algorithm is widely used in robot path planning. However, the classic ant colony algorithm still has the problems of slow convergence speed and easily fall into the local optimal solution. Therefore, this paper proposes an improved ant colony algorithm. Firstly, the Floyd algorithm is introduced to generate the guidance path to optimize the initial pheromone matrix and effectively accelerate the initial convergence speed of the ant colony algorithm. Ant fallback strategy can help avoid ants dying due to the deadlock dilemma and improve the global search ability of the algorithm. The improved heuristic function proposed by referring to the gravity concept in the APF method accelerates the convergence speed of the ant colony algorithm. It makes up for the influence of the fallback strategy on the convergence rate. The pheromone updating method based on a multi-objective optimization idea and dynamic principle considers the path length, path security, and path energy consumption. It helps the ant colony algorithm avoid the local optimal solution and improves the comprehensive performance of the algorithm, which is more suitable for mobile robots. Connectivity processing and the quadratic B-spline method effectively reduce the redundant nodes of the path, improve the smoothness of the path and further shorten the path length. Through experimental comparisons, as can be seen, the improved algorithm has strong stability. From the simple obstacle environment to the complex obstacle environment, it can always maintain the optimal comprehensive performance, the shortest path, and the least corner. The problems of the classic ant colony algorithm has been solved. In addition, the multi-objective optimization idea and the node optimization method introduced in the improved algorithm can effectively help the mobile robot to save energy and improve the work efficiency.",
"introduction": "Introduction The path planning of mobile robot is to plan the optimal path from the starting point to the target point in the specified area (Chen et al., 2020 ). At present, path planning algorithms is mainly presented in the form of traditional algorithms and intelligent algorithms. The traditional algorithms include the A * Algorithm (Xiong et al., 2020 ), Tabu Search (TS) (Khaksar et al., 2012 ), and D * Algorithm (Yao et al., 2021 ), etc. The intelligent algorithms include Ant Colony Optimization (ACO) (Wang, 2020 ), Particle Swarm Optimization (PSO) (Wang et al., 2020a ), Genetic Algorithm (GA) (Chen and Gao, 2020 ), etc. Intelligent algorithms can also be subdivided. Among them, the ant colony algorithm and particle swarm optimization algorithm belong to the swarm intelligent algorithm. Swarm intelligent algorithm has been a hot spot in path planning. There are two modes of swarm intelligence, namely, ant colony algorithm and particle swarm optimization algorithm. Swarm intelligence mainly refers to the intelligent behavior of many non-intelligent individuals in a group through simple cooperation. Swarm intelligence is applied to path planning, taking the ant colony algorithm as an example. It shows that a single ant in the ant colony has no intelligence, but through cooperation to form a complete system, it evolves into an intelligent whole that can explore the optimal path in a complex environment. Therefore, it is widely studied and applied in path planning. Swarm intelligence is mainly manifested in five principles: (1) Proximity principle; (2) Quality principle; (3) The principle of diverse response; (4) Stability principle; (5) Adaptability principle. Swarm intelligence also has four features. (1) The control of swarm intelligence is decentralized, and there is no unified control center, so it can adapt to various environments and has strong robustness. For example, the ant colony algorithm can carry out path planning in various complex environments and obtain the optimal path. (2) Each individual in the swarm can communicate by changing the environment, which has good scalability. For example, the ants change the pheromone content in the environment by leaving pheromones on the path, to realize communication with other individuals. (3) The behavior of individuals in the swarm or the rules they follow are very concise, so it is very convenient to realize swarm intelligence. For example, individuals in the ant colony only need to follow the state transition rules to find the path and leave pheromones to inform the latecomers. (4) The complex behavior of a swarm is the result of individual communication and cooperation. Under the guidance of appropriate rules, swarm intelligence can play a role in some form of emergence through communication and cooperation. For example, individuals in the ant colony interact through pheromones and then complete path exploration. Then pheromone update mechanism plays a role in guiding the ant colony to optimize the path further and finally get the optimal path. Ant colony algorithm in swarm intelligence fully reflects the characteristics of swarm intelligence. It is simple to set parameters, suitable for various complex environments, and has strong robustness. Therefore, it is widely used in robot path planning. In this paper, the ant colony algorithm will be deeply studied and optimized. Italian scholar Marco Dorigo proposed the ant colony algorithm in 1992. The algorithm was derived from the path finding behavior of ants looking for food sources in nature (Mac et al., 2016 ). The most prominent feature of the ant colony algorithm is the positive feedback mechanism (Zhang et al., 2021 ) which is conducive to obtaining the optimal solution quickly. Then, the ant colony can change the environment by releasing pheromone, so as to communicate indirectly (Yi et al., 2019 ). At last, the ant colony adopts the distributed computing method to search the path (Zheng et al., 2020 ), and the parallel computing is carried out by multiple individuals at the same time. Nevertheless, the defects of slow convergence speed and easy to fall into the local optimal solution cannot be ignored (Yang et al., 2019 ). For the defects of the ant colony algorithm, many researchers have proposed optimization schemes that can be divided into three categories. (1) In consideration of the slow convergence speed of the ant colony algorithm, improve the initial pheromone allocation method, or improve the state transition probability matrix, such as Luo et al. ( 2020 ) and Li et al. ( 2021 ); etc. (2) In order to optimize the defect of the ant colony algorithm that it is easy to fall into local optimal solution, the pheromone matrix updating method is optimized or pheromone concentration is limited, such as Akka and Khaber ( 2018 ) and Wang et al. (2020), etc. (3) Many schemes to improve the path smoothness of ant colony algorithm have been proposed. There are mainly two ways: improving the heuristic function and optimizing the path nodes, such as Dai et al. ( 2019 ) and Yang et al. ( 2019 ), etc. Some optimization schemes will be introduced in detail below. To improve the ant colony algorithm, there are a lot of optimization schemes (Akka and Khaber, 2018 ; Luo et al., 2020 ; Li et al., 2021 ). In Luo et al. ( 2020 ), an improved ant colony algorithm was proposed. The algorithm constructs unequally distributed initial pheromone in the early stage of path planning. At the same time, the pseudo-random state transition rule is used to select the trail. The deficiency is that the algorithm only sets the initial pheromone according to the position information of the node, which is not conducive to avoiding obstacles in the process of the ant search path, and the guidance of the ant colony is not direct enough. In Li et al. ( 2021 ), an improved algorithm based on turning angle constraint was proposed. Firstly, the initial pheromone concentration between the starting node and the target node is increased. Then, the evaluation function and rotation constraint factor of the A * algorithm is added to the heuristic function. The nodes with the optimal path length and rotation number can be selected in the next step. Finally, in the pheromone updating part, the distribution principle of the wolf swarm algorithm is introduced to strengthen the influence of a high-quality population. The algorithm proposed by Li effectively avoids falling into optimal local solutions, but the convergence speed in a complex environment cannot meet the requirements. In Akka and Khaber ( 2018 ), an improved ant colony optimization algorithm was proposed. The algorithm uses stimulus probability to help ants select the following grid, and uses new heuristic information to improve visibility accuracy. In addition, the improved algorithm adopts new pheromone updating rules and dynamically adjusts the evaporation rate, which accelerates the convergence speed and expands the search space. This algorithm does not consider the requirements of path smoothness when effectively accelerating the convergence speed, which is not conducive to reducing the energy consumption and mechanical loss of the robot. In summary, to solve the problems of slow convergence rate and easily fall into the local optimal solution of ant colony algorithm, this paper proposes an improved algorithm. (1) For the difficulties in Luo et al. ( 2020 ), the Floyd algorithm is introduced to generate the guidance path. The path is a feasible path without collision with obstacles. Setting the initial pheromone based on the track can help the ant colony avoid blind search and take into account the obstacle avoidance needs. (2) Considering that the ants easily fall into the deadlock and self-locking state, the fallback strategy is proposed to reduce the number of dead ants and help improve the success rate of the algorithm to solve the way. (3) For the problems that have not been solved in Li et al. ( 2021 ), the APF method and the concept of the distance between the optional node and the target node are introduced to optimize the structure of the heuristic function, which improves the state transition probability and accelerates the convergence rate. (4) Given the shortcomings of Akka and Khaber ( 2018 ), the connectivity principle and quadratic B-spline curve optimization method are proposed to optimize the corner nodes, further shortening the path length and reducing the mechanical loss of the robot in the working process. (5) Moreover, this paper proposes a multi-objective optimization method, taking into account the path length, path safety, and path energy consumption, to solve the bearing with the highest comprehensive quality. The pheromone updating method is improved based on the multi-objective optimization method and dynamic principle, which prevents the algorithm from falling into the local optimal solution to the greatest extent. The rest of this paper is as follows. The second part briefly describes the two-dimensional grid environment modeling method, which is a crucial environment for algorithm operation. The third part introduces the core part of the classic ant colony algorithm. The fourth part gives the progress measures of the algorithm in detail. In the fifth part, the classic ant colony algorithm and the improved algorithm are compared and analyzed. The sixth part summarizes the contributions and shortcomings of the improved algorithm, and briefly looks forward to future work.",
"discussion": "Experimental results and discussions In this section, the effectiveness of the improved algorithm in path planning is verified through different scenarios. All experiments were performed using the same PC. The MATLAB (R2016b) programming platform was used to encode and implement all algorithms. In order to obtain real experimental results and avoid accidental situations, all experiments were carried out independently under the same experimental conditions. The 26 × 26 scale grid map is adopted in this paper. There are three different environments, namely, the concentrated obstacle environment, the partially dispersed obstacle environment, and the decentralized obstacle environment. The algorithm in this paper, the classic ant colony algorithm, and the algorithm of Li et al. ( 2021 ), Luo et al. ( 2020 ), and Akka and Khaber ( 2018 ) are compared experimentally. The algorithm parameters are set as shown in Table 2 . Table 2 Parameter setting. \n Parameter \n Starting point S 1 Target point E 676 Maximum number of iterations K 100 The number of ants M 50 Pheromone heuristic factor α 1 Expected heuristic factor β 6 Pheromone volatilization factor ρ 0.6 Pheromone intensity factor Q 1 Pheromone penalty evaporation coefficient λ 15 Concentrated obstacle environment In the concentrated obstacle environment with the 26 × 26 scale grid, the experimental results of five algorithms are shown in Figure 10 . Figure 10 Experimental results of three algorithms in the concentrated obstacle environment. (A) Classic ant colony algorithm, Luo et al. ( 2020 ); Li et al. ( 2021 ), and Akka and Khaber ( 2018 ) (B) Improved algorithm and comparison of five algorithms. The specific results of the experiment are shown in Table 3 . Index 1 is the average path length, index 2 is the optimal path length, index 3 is the average number of iterations, and index 4 is the average number of corners. Table 3 Comparison of five algorithms. \n Index \n \n Concentrated obstacle \n \n Partially decentralized \n \n Decentralized obstacle \n \n environment \n \n obstacle environment \n \n environment \n Classic ACO 1 39.2843 41.4578 43.2763 2 38.8701 40.0416 41.4558 3 70 65 75 4 12 14 18 Li et al. ( 2021 ) 1 38.5772 39.9771 48.6639 2 38.2843 39.1127 46.2543 3 5 10 18 4 9 12 15 Luo et al. ( 2020 ) 1 41.5772 40.4056 43.2132 2 40.4807 39.6985 41.7990 3 12 9 8 4 15 15 16 Akka and Khaber ( 2018 ) 1 38.3045 40.8078 41.3356 2 37.9793 39.6853 40.9214 3 10 10 12 4 10 13 16 Improved algorithm 1 37.5438 39.0204 39.6872 2 37.2033 38.9281 39.1280 3 7 11 10 4 7 9 11 It can be seen from Figure 10 and Table 3 that the comprehensive performance of the improved algorithm in this paper is the best in the concentrated obstacle environment. In terms of the optimal path length, the improved algorithm is 4.29% less than the classic ant colony algorithm, 2.82% less than the algorithm in Li et al. ( 2021 ), 8.10% less than the algorithm in Luo et al. ( 2020 ), and 2.04% less than the algorithm in Akka and Khaber ( 2018 ). In terms of the average path length, the improved algorithm is 4.43, 2.68, 9.70, and 1.99% less than other algorithms, respectively. In terms of the average number of iterations, the improved algorithm is 63 times less, 2 times more, 5 times less and 3 times less than other algorithms, respectively. In terms of the average number of corners, the improved algorithm is 41.67, 22.22, 53.33, and 30% less than other algorithms respectively. To sum up, in the concentrated obstacle environment, the performance of the improved algorithm in this paper is better than the other four algorithms, including the classic algorithm. Partially decentralized obstacle environment In the partially decentralized obstacle environment with the 26 × 26 scale grid, the experimental results of five algorithms are shown in Figure 11 . Figure 11 Experimental results of three algorithms in the partially decentralized obstacle environment. (A) Classic ant colony algorithm, Luo et al. ( 2020 ); Li et al. ( 2021 ), and Akka and Khaber ( 2018 ). (B) Improved algorithm and comparison of five algorithms. The specific results of the experiment are shown in Table 3 . As can be seen from Figure 11 and Table 3 , the performance of the improved algorithm in this paper is still better than that of other algorithms in the partially decentralized obstacle environment. In terms of the optimal path length, the improved algorithm is 2.87% less than the classic ant colony algorithm, 0.47% less than the algorithm in Li et al. ( 2021 ), 1.94% less than the algorithm in Luo et al. ( 2020 ), and 1.91% less than the algorithm in Akka and Khaber ( 2018 ). In terms of the average path length, the improved algorithm is 5.88, 2.39, 3.43, and 4.38% less than other algorithms respectively. In terms of the average number of iterations, the improved algorithm is 54 times less, 1 time more, 2 times more and 1 time more than other algorithms respectively. In terms of the average number of corners, the improved algorithm is 35.71, 25, 40, and 30.77% less than other algorithms respectively. It can be seen from the above that the performance of the improved algorithm in this paper still has certain advantages in the partially decentralized obstacle environment. Decentralized obstacle environment In the decentralized obstacle environment with the 26×26 scale grid, the experimental results of five algorithms are shown in Figure 12 . The specific results of the experiment are shown in Table 3 . Figure 12 Experimental results of three algorithms in the decentralized obstacle environment. (A) Classic ant colony algorithm, Luo et al. ( 2020 ); Li et al. ( 2021 ), and Akka and Khaber ( 2018 ). (B) Improved algorithm and comparison of five algorithms. It can be seen from Figure 12 and Table 3 that the improved algorithm in this paper has more obvious advantages than other algorithms in the decentralized obstacle environment. In terms of the optimal path length, the improved algorithm is 5.62% less than the classic ant colony algorithm, 15.41% less than the algorithm in Li et al. ( 2021 ), 6.39% less than the algorithm in Luo et al. ( 2020 ), and 4.38% less than the algorithm in Akka and Khaber ( 2018 ). In terms of the average path length, the improved algorithm is 8.29, 18.45, 8.16, and 3.99% less than other algorithms, respectively. In terms of the average number of iterations, the improved algorithm is 65 times less, 8 times less, 2 times more and 2 times less than other algorithms, respectively. In terms of the average number of corners, the improved algorithm is 38.89, 26.67, 31.25, and 31.25% less than other algorithms, respectively. From the above comparisons, as the complexity of the environment increases, the improved algorithm in this paper always has significant advantages. From the above experiments, it can be seen that in the simple environment, except for the classic ant colony algorithm, the other three algorithms are close to the improved algorithm. As the complexity of the environment increases, the indicators of the five algorithms have changed, and the performance of the improved algorithm has always remained stable, which has been better than the other four algorithms, including the classic algorithm. Among the five algorithms, the improved algorithm is the best, which is most conducive to the energy-saving and stable operation of the robot."
} | 5,056 |
35492914 | PMC9050346 | pmc | 6,162 | {
"abstract": "This study explored the optimum conditions to achieve superhydrophobicity in polyethylene terephthalate (PET) in terms of crystallinity and microstructure. Surface superhydrophobicity was achieved by nanostructures induced by oxygen plasma etching and the recovery process of low surface energy through thermal aging of various PETs; semi-crystalline biaxial PET (B-PET) film, amorphous PET (A-PET) film, and semi-crystalline PET (F-PET) fabric. Under the anisotropic plasma etching, the nanostructures on the B-PET film were the longest, followed by the F-PET fabric, which developed a hierarchical micro/nanostructure, then the A-PET film. During thermal aging at 80 °C near T g , the plasma-treated A-PET film recovered its superhydrophobicity within 3 h, while the plasma-treated B-PET film did not exhibit superhydrophobicity. At 130 °C, higher than T g , the plasma-treated B-PET film recovered its superhydrophobicity within 1 h, but the plasma-treated A-PET film became opaque as its nanostructures deformed, decreasing its superhydrophobicity. The plasma-treated F-PET fabric exhibited faster recovery and greater superhydrophobicity than the plasma-treated B-PET film, due to its hierarchical micro/nanostructure. In addition, hydrophobic recovery during thermal aging was proved with a decrease in surface polar groups, lowering the surface energy using XPS analysis. Therefore, by designing the ratio of crystal to amorphous regions and surface micro/nanostructures, one can rapidly fabricate superhydrophobic PETs without additional surface finishing.",
"conclusion": "Conclusions The rapid achievement of superhydrophobicity was explored in three different PET materials through plasma-induced nanostructuring and subsequent thermal aging, which are the main processes for affecting the hydrophobic recovery and environment/human friendly methods. Various oxygen plasma etching durations and thermal aging conditions were applied to each specimen, and the degree of hydrophobic recovery was compared by measuring the static CAs and SAs. Observation of nanostructure formation by oxygen plasma etching revealed that the B-PET film, similar to the F-PET fabric, formed nanostructures more rapidly than the A-PET film. This suggests that the nanostructuring rate in oxygen plasma etching is strongly affected by the crystallinity of PET materials as crystalline regions promote selective plasma etching. After oxygen plasma etching, the A-PET film effectively recovered its superhydrophobicity when thermally aged near its T g (80 °C). However, B-PET film and F-PET fabric having a higher degree of crystallinity were required a higher temperature (130 °C) than the T g to achieve superhydrophobicity, since the crystal regions hinder chain mobility. In the case of the A-PET film, when thermally aged at 130 °C, superhydrophobicity was reached within 1 h, but after 1 h, the nanostructures on the A-PET film deformed and decreasing superhydrophobicity due to less thermal stability for nanostructures. Therefore, with respect to superhydrophobic robustness, some degree of crystallinity could be necessary, because the PET material with some degree of crystallinity forms rapidly and has sturdy nanostructures. Compared to the B-PET film, F-PET fabric showed a faster and greater superhydrophobicity because of its hierarchical structure of micro- and nano-roughness. The intensive XPS examination of the surface characteristics of PETs before and after oxygen plasma etching or thermal aging showed that, regardless of samples, hydrophobic recovery is also led by a decrease in the surface energy as the polar components introduced by oxygen plasma etching were rearranged into the bulk. From these results, when the PET materials, and possibly other polymers, have moderately high crystallinity, nano-roughness can be achieved rapidly with a high aspect ratio by oxygen plasma etching and superhydrophobic recovery can be robustly achieved with subsequent thermal aging and no additional chemical coating. From these results, we assume that these findings could be widely applicable for functional clothing textiles and biomedical goods, as well as many other products made of polymeric materials.",
"introduction": "Introduction A superhydrophobic surface, as observed naturally in the lotus leaf or water strider, is defined as a surface with a static water contact angle (CA) of >150° and a shedding angle (SA) of <10°. This surface has various functional properties, for instance, water and oil resistance, anti-fogging, anti-freezing, and self-cleaning for water and dirt. In particular, superhydrophobic textiles can be used as functional materials to reduce the frequency of necessary washing and, therefore, provide easy care of textiles. Owing to these advantages, many studies have been conducted on the implementation of superhydrophobic surfaces into various materials requiring breathable, anti-staining, and antibacterial functions. It is known that a superhydrophobic surface can be fabricated from hierarchical nano- and microstructures with a low-surface-energy coating. Therefore, superhydrophobic surfaces with hierarchical structures have been made by attaching nanoparticles such as SiO 2 , 1 TiO 2 , 2 and carbon nanotubes (CNT) 3 onto inherently microscale fabrics and coating them with low surface energy materials, or by dispersing nanoparticles in the low surface energy materials and then applying them to fabrics. However, attached nanostructures are easily removed from fabric surfaces during daily use or washing because of their low adhesion with fabric surfaces, and their byproducts have been associated with harmful effects on biological systems in the environment or human body. 4 As for low surface energy coating materials, fluoro compounds containing perfluoroalkyl groups have been most commonly utilized since they can create not only water repellency, but also oil resistance. 5 However, fluoro compounds generate carcinogens such as perfluorooctanesulfonic acid or perfluorooctanoic acid during decomposition, which are not easily biodegradable and remain in the human body and environment with harmful effects. 6–8 Therefore, international non-governmental environmental organizations (Greenpeace) and researchers have actively informed the public of the risks of using fluoro compounds and tried to restrict and regulate their use, reporting that methods to replace fluoro compounds are required. Consequently, coating materials for direct contact with humans need to be developed without fluoro compounds. Recent studies have reported a superhydrophobic surface, which is expected to be friendly to the human body and environment, produced by controlling surface energy through thermal aging without using any additional chemicals or coating. Oh et al. 9,10 suggested fabricating the superhydrophobic surface using a nanostructuring technique based on oxygen plasma etching or alkaline hydrolysis and the reduction of surface energy with a non-finishing coating, thermal aging. They showed excellent self-cleaning properties with static CAs of >170° and SAs of <10°. These studies were merely focused on the effect of temperature on the recovery of hydrophobicity from the plasma-treated or alkaline hydrolyzed hydrophilic PET surface, which is an external factor. Consequently, it was discovered that the recovery rate of hydrophobicity in polymeric materials is affected by their intrinsic crystallinity and molecular structure, as well as by the fabrication process. Therefore, in this study, we observed the effect of crystallinity of PET materials on the formation of nanostructures and the rate of subsequent hydrophobic recovery, suggesting the optimum condition to exhibit superhydrophobicity on the plain film or fabric made of microfibers. The materials for this study consist of semi-crystalline biaxial PET (B-PET) film, amorphous PET (A-PET) film, and semi-crystalline microstructured PET (F-PET) fabric. The effects of the crystallinity of the specimen were observed on the formation of nanostructures on each surface by oxygen plasma etching. Subsequently, the hydrophilic PET materials were restored to hydrophobicity or further rendered to superhydrophobicity through thermal aging, in which the effect of crystallinity was in detail analyzed at temperatures under, near, and above the glass transition temperature ( T g ). The hydrophobic recovery caused by thermal aging was explored by measurement of the water contact angle (CA) and shedding angle (SA). Surface roughness as well as chemical composition were analyzed with respect to the above temperatures using XPS analysis. Consequently, advantageous and efficient conditions for developing practical superhydrophobic materials were proposed for wider applications.",
"discussion": "Results and discussion Nanostructuring rate with plasma etching time With increasing oxygen plasma etching time, the nanostructures created by the selective plasma etching mechanism 17 were formed on the flat surface of every specimen regardless of crystallinity. In general, nanobumps were generated first at the beginning of etching, then the morphology evolved into nanopillars and nanohairs as the etching duration increased ( Fig. 2 and ESI Fig. 1–6 † ). It has been suggested that the Fe or Cr components in the stainless-steel cathode were sputtered and codeposited on the sample surface as the plasma radical ions formed during glow discharge bombarded the cathode plate. Then the metal clusters by diffusion to form a self-etching mask, which prevents the chemical reaction between oxygen plasma radicals and PET at the surface. In contrast, in the specimen areas with no metallic clusters, rapid surface etching occurs, resulting in anisotropic etching. This causes differences in etching speed depending on the position on the specimen surface during oxygen plasma treatment, and the morphology of nanostructures become gradually longer with plasma treatment duration ( Fig. 2a–f ). 18 Fig. 2 Tilted SEM images of B-PET film (a and d), A-PET film (b and e) and F-PET fabric (c and f) for plasma etching durations of 5 and 15 min (×150000, scale bar: 200 nm). Insets are the low magnification images of each sample (×200, scale bar: 100 μm). The diameter (g), distance (h), length (i), and aspect ratio (j) of each PET material measured with oxygen plasma etching duration. At the beginning of plasma etching, the specimens did not show differences in the diameters and lengths of nanostructures, but the lengths of the nanostructures varied as oxygen plasma etching time increased ( Fig. 2g and i ). The nanostructuring rates were compared between specimens through the measurement of nanostructure length ( Fig. 2i ). The nanostructures on the B-PET film were the longest and its nanostructuring rate was the highest, followed by the F-PET fabric and A-PET film, respectively. This reason for this stems from their difference in crystallinity. The B-PET film or F-PET fabric have a three-phase composition of amorphous, ordered amorphous, and crystalline phases, 19 whereas the A-PET film has a single amorphous phase ( Table 1 ). The amorphous regions were etched more uniformly and easily, with a constant and fast etching rate, across the specimens compared with the crystalline regions that have a highly ordered crystal structure and high bond energy between the polymer chains resulting in slower etching. 19 Consequently, after plasma etching, the A-PET film had a larger portion of surface uniformly removed by plasma etching compared to the B-PET film and F-PET fabric. 20,21 As a result, the A-PET film became thinner, and its nanoscale surface roughness decreased. 20 Therefore, it was hypothesized that the anisotropic etching mechanism relies on the crystallinity of the sample as well as on codeposited metal elements on the surface due to the high etching rate of the amorphous region, which surpasses the mask forming rate, making it difficult to form clusters or etching masks there. Therefore, the A-PET film shows a significantly slower surface to obtain nanostructures with an aspect ratio of 3 or higher ( Fig. 2b, e and j ). However, the B-PET film and F-PET fabric showed well-developed nanostructures with high aspect ratios ( Fig. 2a, c, d and f ), even after a short period of time, since they have a crystalline region that can act as a deposition site for metal elements. For example, nanostructures with an aspect ratio of 3 or higher were formed within 5 min etching on the B-PET film ( Fig. 2j ). These results suggest that the nanostructuring rate in oxygen plasma etching is strongly affected by the crystallinity of PET, 19 as the crystalline region promotes selective plasma etching. 22 It was also found that even though the B-PET film and F-PET fabric have the same chemical compositions and similar thicknesses (0.2 ± 0.05) and crystallinity (37.0% and 36.5%) ( Table 1 ), the B-PET film has a higher nanostructuring rate than the F-PET fabric. This could be because the F-PET fabric has inherent micro-roughness as it is composed of fibers, yarns, and pores ( Fig. 2c ). Thus, we assume that the etching masks formed by the deposition of metal clusters would not be as regular as those on the B-PET film, resulting in a lower nanostructuring rate. The change in crystallinity for each specimen was measured before and after oxygen plasma etching and ranged from 0.93% to 2.88% for the A-PET film, from 37.0% to 36.8% for the B-PET film, and 36.5% to 36.2% for the F-PET fabric. These results were corresponded with the XRD results as shown in ESI Fig. 7a and b. † Untreated B-PET film showed a strong peak at 2 Θ = 26.16° and oxygen plasma etched B-PET had no difference with the peak of untreated B-PET. On the other hand, untreated A-PET film showed 0% crystallinity and the amorphous halo which is a typical pattern of amorphous materials 23–27 and the halo area was almost same after oxygen plasma etching. Thus, the crystallinity did not change significantly during the treatment, indicating that oxygen plasma etching has no significant effect on the entire specimen since it only affects the surface region, up to approximately several hundred nanometers deep. 28,29 Recovery of surface hydrophobicity of B-PET film and A-PET film The effects of crystallinity on hydrophobic recovery by thermal aging in nanostructured PET samples were explored. It is known that after surface modification or nanostructuring by oxygen plasma etching, any surface, including on polymeric materials, will be hydrophilic as the surface energy is increased due to free radical or polar groups generated by the oxygen plasma. As the plasma-treated surfaces are exposed to air or thermal condition, the surface energy will be reduced and the water contact angle will increase over time and eventually revert to the original hydrophobic state of the PET material. 9 Therefore, hydrophobic recovery was explored on the surfaces after oxygen plasma etching. The static CAs and SAs of two different samples of the 10 min plasma-etched B-PET film and 10 and 20 min plasma-etched A-PET film were measured ( Fig. 3 , Tables 2 and 3 ). The PET films with and without nanostructures were treated for 24 h at 40 °C, 80 °C, and 130 °C. Fig. 3a and Table 2 show the static CAs and SAs of the B-PET film that was plasma etched for 10 min and then thermally aged. After thermal aging for 24 h at 40 °C, which is lower than its T g , the static CA was below 40° and the SA was higher than 45° because the PET films would not change its chemical phase or molecular arrangement, and thus retained hydrophilicity at 40 °C. By contrast, the B-PET film thermally aged at 80 °C, near its T g , exhibited a sudden increase in hydrophobicity between 1 and 3 h of treatment, with a final static CA of 143.4° ± 1.4° and SA of 18.7° ± 16.1° after 24 h. However, during thermal aging at 130 °C, which is higher than its T g , it showed a rapid recovery in superhydrophobicity. After thermal aging for 1 h at 130 °C, the B-PET film exhibited a sharp increase in its static CA to 146.9° ± 3.9° and decrease in SA to approximately 4.8° ± 0.8°. After 3 h, both static CA and SA displayed the superhydrophobicity condition and after 24 h changed to 170.0° ± 2.7° and 0.0° ± 0.0°, respectively ( Fig. 3a and Table 2 ). This shows that the higher thermal aging temperature lead fast and higher hydrophobic recovery. In both 10 min and 20 min plasma-stabilize and increased continuously even after thermal aging for 24 h, meaning that hydrophobic recovery was not complete yet. The 10 min plasma-etched B-PET sample thermally aged at 80 °C showed the superhydrophobicity condition and after 24 h changed to 170.0° ± 2.7° and 0.0° ± 0.0°, respectively ( Fig. 3a and Table 2 ). This hydrophobicity in all cases whereas the 20 min plasma-etched A-PET film aged at 80 °C showed high hydrophobicity with a static CA of 180.0° ± 0.0° and a SA of 0.0° ± 0.0° after 5 h, thus exhibiting superhydrophobicity. However, at 130 °C, the static CA of the 10 min plasma-etched A-PET film reached its maximum after 1 h and then gradually decreased over remaining aging duration. Furthermore, the 20 min plasma-etched A-PET film showed its maximum superhydrophobicity after 1 h of thermal aging with a static CA of 164.6° ± 4.1° and a SA of 2.0° ± 0.0°, but with further aging, the static CA drastically decreased to 110.0° ± 1.9° and the SA increased to over 45°. Fig. 3 Static contact angles of (a) untreated (UT) and 10 min plasma-etched B-PET film, and (b) untreated (UT), 10 min, and 20 min plasma-etched A-PET film depending on thermal aging temperatures at room temperature (RT), 40, 80 and 130 °C with aging duration. Shedding angles of 10 min plasma-etched B-PET film depending on thermal aging temperature and treatment duration B-PET film Etching duration (min) 10 Thermal aging time (h) Temp. (°C) 40 80 130 Shedding angle (°) 1 >45 >45 4.8 ± 0.8 3 >45 >45 1.6 ± 0.8 5 >45 >45 0.0 ± 0.0 9 >45 >45 0.0 ± 0.0 15 >45 23.5 ± 13.1 0.0 ± 0.0 24 >45 18.7 ± 16.2 0.0 ± 0.0 Shedding angles of 10 min and 20 min plasma-etched A-PET film depending on thermal aging temperature and treatment duration A-PET film Etching duration (min) 10 20 Thermal aging time (h) Temp. (°C) 40 80 130 40 80 130 Shedding angle (°) 1 >45 >45 >45 >45 23.0 ± 24.1 0.0 ± 0.0 3 >45 >45 >45 >45 0.3 ± 0.6 2.0 ± 0.0 5 >45 >45 >45 >45 0.0 ± 0.0 >45 9 >45 >45 >45 >45 0.0 ± 0.0 >45 15 >45 >45 >45 >45 0.0 ± 0.0 >45 24 >45 >45 >45 >45 0.0 ± 0.0 >45 When the surface nanostructures of the A-PET film were examined with respect to aging temperature, it was found that the morphology was mostly unchanged when thermally aged at 80 °C even after 24 h as the R a and R q values remained similar at approximately 100 and 77 nm, respectively (ESI Fig. 8c and d † ). This confirmed that thermal aging near T g does not affect the morphology of surface nanostructures. Furthermore, it did not affect the crystallinity or transparency of the A-PET film, resulting in the recovery of superhydrophobicity. On the other hand, the A-PET specimens that underwent thermal aging at 130 °C, above T g , became opaque and showed decreased hydrophobicity, making the flat-water droplet on the surface ( Fig. 4a ). This observation revealed that in this condition the nanostructures mostly lay down or disappear as shown in Fig. 4d and g . This is because A-PET film with no crystallinity has low physical and dimensional stability, 30 and thus, when it is thermally aged at 130 °C, the Young's modulus decreases until the material becomes rubbery, causing the surface nanostructures morphology to be altered ( Fig. 4b–g ). 31 Additionally, after thermal aging at 130 °C, above the crystallization temperature ( T c , approximately 125.3 °C), crystallinity increased from 0% to 20% as heat-induced spherulitic crystals were generated in the bulk of the A-PET film regardless of oxygen plasma etching. 20,32 Therefore, as shown in ESI Fig. 7b † the XRD patterns were changed after thermal aging and crystallinities measured by XRD analysis were changed from 0% to 22.2%, which is similar with the DSC result. It was suggested that for amorphous polymers, thermal aging at a temperature near T g causes molecular chain rearrangement, moving polar components into the bulk and arranging the non-polar components on the surface which lowers the surface energy 9,33 and restores a high surface hydrophobicity. Therefore, for PET materials with low crystallinity, thermal aging at a temperature near or below T g is more desirable for realizing superhydrophobicity than a temperature above T g . Hydrophobic recovery behavior for the 10 min plasma-etched B-PET film and the 20 min plasma-etched A-PET film were compared since both have nanostructures with similar geometries of diameters, lengths, and spacings. After oxygen plasma etching and thermal aging at 80 °C for 5 h, the 20 min plasma-etched A-PET film had a static CA of 180.0° ± 0.0° and a shedding angle of 0.0° ± 0.0° ( Fig. 3b and Table 3 ), exhibiting a greater and faster surface hydrophobic recovery than that of the 10 min plasma-etched B-PET film, which showed a static CA of 143.3° ± 1.4° and a shedding angle of 18.7° ± 6.1° ( Fig. 3a and Table 2 ). Furthermore, the time for the A-PET film to reach its maximum static CA was shorter than that for the B-PET film. 34 The reason for this is that in the densely packed crystalline region, molecular chain movement is limited as rotational and translational movement of the polymer chains is inhibited, 9,33 whereas molecular chain movement is freer in the amorphous region. 35 Consistently, during thermal aging at 130 °C A-PET film tended to show greater superhydrophobicity more quickly, within 1 h, than the B-PET film. However, as thermal aging continued, the nanostructures on the A-PET film lay down or melted, causing a decrease in superhydrophobicity as discussed above. On the other hand, the B-PET film could maintain it superhydrophobicity without changing its nanostructures ( Fig. 4a ). From these results, it can be determined that if crystallinity is high in the PET film, the static CA recovery rate is slow so it takes longer to reach the maximum static CA, but in terms of the robustness under continuous thermal processing and speed of nanostructure formation, a crystal region would be a better condition to exhibit superhydrophobicity. Fig. 4 A photograph of 20 min plasma-etched B-PET and A-PET films before and after thermal aging at 80 and 130 °C for 24 h (a). SEM images of 10 min plasma-etched A-PET film before (b) and after thermal aging at 80 °C (c) and 130 °C (d) for 24 h and 20 min plasma-etched A-PET film before (e) and after thermal aging at 80 °C (f) and 130 °C (g) for 24 h. Recovery of surface hydrophobicity for B-PET film and F-PET fabric During oxygen plasma etching, a nanoscale roughness was formed on the B-PET film as shown in Fig. 2a , while a hierarchical structure was developed on the F-PET fabric where fibers several tens of micrometers in length have nanohairy structures on its surfaces ( Fig. 2c ). Therefore, in order to investigate the effect of micro roughness on thermal aging, the 10 min plasma-etched B-PET film and the 15 min plasma-etched F-PET film were compared as shown in Fig. 3a and 5 . Specimens were chosen to have similar nanostructure morphology for the diameter, length, and spacing between nanostructures ( Fig. 2g–i ). Before discussing the hydrophobic recovery between B-PET film and F-PET fabric, the surface wettability change on the F-PET was analysed depending on the plasma etching duration or thermal aging. Fig. 5 shows the surface wettability of the 10 min and 15 min plasma-etched F-PET fabric according to the thermal aging temperature and time. None of the F-PET fabrics showed hydrophobic recovery at 40 °C. After thermal aging at 80 °C for 24 h, the static CA increased noticeably, but did not reach equilibrium. When thermal aged at 130 °C, however, superhydrophobicity was achieved. After treated at 130 °C for 1 h, the 15 min plasma-etched F-PET fabric recovered a static CA of 157.5° ± 3.7° and a SA of 0.6° ± 0.5° and after 3 h, it showed extreme superhydrophobicity with a static CA of 180.0° ± 0.0° and SA of 0.0° ± 0.0° ( Fig. 5 and Table 4 ). In order to illustrate the effect of micro-etched F-PET fabric. After thermal aging performed at 80 °C for 24 h, the 10 min plasma-etched B-PET film showed a static CA of 143.4° ± 1.4° and a SA of 18.7° ± 16.2°, exhibiting higher surface hydrophobic recovery than that of the 15 min plasma-etched F-PET fabric which showed a static CA of 56.4° ± 67.5° and a SA of >45°. Considering that the 10 min plasma-etched B-PET film and the 15 min plasma-etched F-PET fabric showed similar levels of crystallinities and nano-roughness before and after thermal aging (ESI Fig. 7a, c, 8a and b † ), this difference in hydrophobic recovery must be due to the difference in polymer orientation caused by different production processes. In other words, this study used B-PET film produced not by simultaneous biaxial stretching 35 but sequential biaxial stretching. According to the X-ray diffraction (XRD) graph ( Fig. 6 ), B-PET film showed a narrow peak at 2 Θ = 26.16°, indicating that the crystal structure is formed only in (100) planes 36 and is highly oriented. Contrarily, for F-PET fabric, which is produced by spinning and drawing, the molecular chains are arranged in the axial direction of fibers, and the peaks were detected at 2 Θ = 17.65°, 22.60° and 25.42°. Thus, the crystals exist in the (010), (−101), and (100) planes. 37,38 Moreover, it was found that the orientation was weaker than in the B-PET film when the peak widths were compared. Therefore, the movement of molecular chains in the B-PET film, which are highly oriented in one plane is easier than in the F-PET fabric which is less oriented in multiple planes. 39 Consequently, the B-PET fabric shows a higher surface hydrophobicity recovery after thermal aging at 40 °C and 80 °C for 24 h than the F-PET fabric. After thermal aging at 130 °C for 24 h, the 10 min plasma-etched B-PET film had a static CA of 170.0° ± 2.7° and a SA of 0.0° ± 0.0° and the 15 min plasma-etched F-PET fabric had a static CA of 180.0° ± 0.0° and a SA of 0.0° ± 0.0°. It was shown that both had greater hydrophobicity recovery at this temperature than at temperatures below and near T g ( Fig. 3a , 5 and Tables 2 and 4 ), which is the temperature at which hard polymers begin to become soft like rubber 40,41 and molecular chain rearrangement occurs. Moreover, when the micro-roughness effect of F-PET fabric was added, higher superhydrophobicity was manifested. As shown in Table 1 , the untreated F-PET fabric showed a static contact angle of approximately 0° due to the inherent micro-roughness of fabric, but only after thermal aging at 130 °C for 24 h, its static contact angle increased to 133.4° ± 1.9° due to the surface molecular chain rearrangement and inherent micro-roughness of the fabric. However, after thermal aging at 130 °C, the original static CA of the B-PET film, which was approximately 82.3° ± 1.4° increased to 98.5° ± 1.8° only by surface molecular chain rearrangement. Additionally, after thermal aging at 130 °C, the SA of B-PET film was 0.0° ± 0.0° from 5 h of thermal aging, whereas that of the F-PET fabric became 0.0° ± 0.0° after 3 h of thermal aging. Thus, even though they have the same chemical structure and similar nanostructure diameters, lengths, and distances of separation, as well as crystallinity, the recovery of superhydrophobicity of the F-PET fabric was faster than that of the B-PET film. This originates from the fact that the F-PET fabric has a small contact area and adhesion force with water drops due to its hierarchical structure. 42,43 On this note, Su et al. 44 mentioned that nano-roughness in hierarchical structures gives a force that can endure high pressures and helps water drops maintain the Cassie–Baxter model because micro-roughness drastically decreases the adhesion area with water droplets. Thus, micro-roughness as well as nano-roughness are favorable for satisfying the superhydrophobic surface condition. The above results imply that micro-roughness enables superhydrophobicity to be achieved with shorter oxygen plasma etching and thermal aging times, which will make it more efficient in reaching superhydrophobicity compared to flat specimens. Fig. 5 Static CAs of untreated, 10 min, and 15 min plasma-etched PET fabric (F-PET) depending on thermal aging temperature and duration. Shedding angles of 10 min and 15 min plasma-etched PET fabric (F-PET) depending on thermal aging temperature and duration F-PET fabric Etching duration (min) 10 15 Thermal aging time (h) Temp. (°C) 40 80 130 40 80 130 Shedding angle (°) 1 >45 >45 6.0 ± 1.9 >45 >45 0.6 ± 0.5 3 >45 >45 3.4 ± 0.9 >45 >45 0.0 ± 0.0 5 >45 >45 3.3 ± 0.6 >45 >45 0.0 ± 0.0 9 >45 >45 3.3 ± 1.2 >45 >45 0.0 ± 0.0 15 >45 >45 3.0 ± 1.0 >45 >45 0.0 ± 0.0 24 >45 >45 1.5 ± 1.0 >45 >45 0.0 ± 0.0 Fig. 6 XRD patterns of untreated biaxial PET (B-PET) film (a) and untreated PET fabric (F-PET) (b). Changes in chemical composition before and after oxygen plasma etching and thermal aging \n Fig. 7 and Table 5 show the surface chemical compositions as determined by XPS in optimum oxygen plasma etching or thermal aging conditions of the B-PET film, A-PET film, and F-PET fabric. Similar peaks were detected on the surfaces of the untreated B-PET film, A-PET film, and F-PET fabric at 285.60 eV, 286.91 eV, 287.41 eV, and 289.58 eV, as seen in Fig. 7a–c , which indicate C–C, C–O, C \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n O, and O C–O bonds, respectively. 45 Based on the bond ratio, the C–C bond from the original molecular structure of the PET shows the highest contribution across all the specimens ( Table 5a–c ). However, after oxygen plasma etching at the optimum condition for each specimen, the number of C–C bonds decreased because of cutting of the surface molecular chain, 46 while the number of C–O, C O, or O C–O bonds increased due to the introduced polar components ( Fig. 7d–f and Table 5d–f ). 9 This suggests that all the specimens become hydrophilic because of the introduced polar groups after oxygen plasma etching. When thermal aging was performed for 24 h at the optimum temperatures of each specimen, the number of C–C bonds increased but the number of C–O, C O, or O C–O bonds decreased ( Fig. 7g–i and Table 5g–i ). It is suggested that the polymer chains were rotated to lower the surface energy during thermal aging. As a result, the polar and non-polar components moved towards the bulk and surface, respectively. 9,10 This indicates that the increase in hydrophobicity from thermal aging after oxygen plasma etching is due to surface molecular chain rearrangement. 33,47 Fig. 7 XPS survey spectra of untreated B-PET film (a), A-PET film (b), and F-PET fabric (c). 10 min plasma-etched B-PET film (d), 20 min plasma-etched A-PET film (e), 15 min plasma-etched F-PET fabric (f), 10 min plasma-etched and thermally aged at 130 °C for 24 h B-PET film (g), 20 min plasma-etched and thermally aged at 80 °C for 24 h A-PET film (h), 15 min plasma-etched and thermally aged at 130 °C for 24 h F-PET fabric (i). Bonding contribution of untreated B-PET film (a), untreated A-PET film (A-PET) (b), untreated F-PET fabric (c), 10 min plasma-etched B-PET film (d), 20 min plasma-etched A-PET film (e), 15 min plasma-etched F-PET fabric (f), 10 min plasma-etched and thermally aged at 130 °C for 24 h B-PET film (g), 20 min plasma-etched and thermal aged at 80 °C for 24 h A-PET film (h), 15 min plasma-etched and thermally aged at 130 °C for 24 h F-PET fabric (i) Bonding contribution (at%) Binding energy (eV) 285.60 286.91 287.41 289.58 Specimens C–C C–O C O O C–O (a) Untreated B-PET film 71.67 5.38 8.02 14.93 (b) Untreated A-PET film 73.36 7.88 9.07 9.69 (c) Untreated F-PET fabric 61.75 13.28 6.74 18.23 (d) B-PET film 10 m 54.01 9.52 21.20 15.27 (e) A-PET film 20 m 58.61 17.92 13.71 9.76 (f) F-PET fabric 15 m 57.45 16.08 7.58 18.89 (g) B-PET film 10 m 130 °C 75.63 6.89 8.32 9.16 (h) A-PET film 20 m 80 °C 72.77 10.63 9.44 7.16 (i) F-PET fabric 15 m 130 °C 76.09 7.73 6.16 10.39"
} | 8,193 |
37763980 | PMC10538107 | pmc | 6,163 | {
"abstract": "Cell growth is inhibited by abiotic stresses during industrial processes, which is a limitation of microbial cell factories. Microbes with robust phenotypes are critical for its maximizing the yield of the target products in industrial biotechnology. Currently, there are several reports on the enhanced production of industrial metabolite through the introduction of Deinococcal genes into host cells, which confers cellular robustness. Deinococcus is known for its unique genetic function thriving in extreme environments such as radiation, UV, and oxidants. In this study, we established that Deinococcus proteolyticus showed greater resistance to oxidation and UV-C than commonly used D. radiodurans . By screening the genomic library of D. proteolyticus , we isolated a gene ( deipr_0871 ) encoding a response regulator, which not only enhanced oxidative stress, but also promoted the growth of the recombinant E. coli strain. The transcription analysis indicated that the heterologous expression of deipr_0871 upregulated oxidative-stress-related genes such as ahp C and sod A, and acetyl-CoA-accumulation-associated genes via sox S regulon. Deipr_0871 was applied to improve the production of the valuable metabolite, poly-3-hydroxybutyrate (PHB), in the synthetic E. coli strain, which lead to the remarkably higher PHB than the control strain. Therefore, the stress tolerance gene from D. proteolyticus should be used in the modification of E. coli for the production of PHB and other biomaterials",
"introduction": "1. Introduction Escherichia coli is one of the most common industrial hosts used as microbial cell factories for producing pharmaceuticals [ 1 ], biopharmaceuticals [ 2 ], fine chemicals [ 3 ], recombinant proteins, and biofuels [ 4 , 5 ]. To maximize the yield of bacterial metabolites, E. coli is subjected to high-cell-density fermentation [ 6 ] under aerobic conditions [ 7 ]. However, the high oxygen concentration and simultaneous accumulation of products such as fuels and chemicals can lead to diverse stresses, especially oxidative stress, which resulted in reduced industrial production [ 8 , 9 ]. Oxidative stress is induced by reactive oxygen species (ROS), such as hydroxyl radical (OH · ), hydrogen peroxide (H 2 O 2 ), and superoxide anion (O 2 − ), through biological respiration including metabolic pathway destruction and DNA alteration [ 10 ]. Therefore, oxidative stress resistance is a critical factor in commercial strains, and various metabolic engineering techniques have been applied to enhance their robustness against oxidative stress [ 11 ]. Engineering regulatory factors, cell membranes, and biosynthetic pathways were employed to improve stress tolerance in E. coli [ 12 ], for example, the heterologous expression of the putative response regulator DRH632 from Antarctic bacteria into E. coli [ 13 ], the random mutation of cAMP receptor protein [ 11 ], and the overexpression of iron exporters, FetA and FetB, into E. coli [ 14 ]. As Deinococcus sp. is known for its ability to survive against multiple stresses such as γ- radiation, UV-C, oxidative stress, and DNA damage reagents, several studies have investigated its genes [ 15 ]. Heterologous expression of Deinococcus genes in E. coli successfully leads to strains with multiple stress resistance. For example, the cold-shock-domain-containing protein PprM [ 16 ], pyrroloquinoline-quinone (PQQ) synthase [ 17 ], manganese (Mn) transporter protein (MntH), and a small heat shock protein (Hsp20) confer oxidative stress tolerance to E. coli [ 18 , 19 ]. Moreover, dr_1558 , a response regulator, confers on E. coli multiple stress resistances to oxidative, acidic, salt, and heat stresses [ 20 ]. Interestingly, some reports suggest that introducing Deinococcal genes into other hosts promotes not only oxidative stress resistance but also its metabolic activity. For instance, the Deinococcal gene irrE ( pprI ) improves the growth and ethanol production of Zymomonas mobilis [ 21 ], the proliferation of E. coli [ 18 ], and lactic acid production in Lactococcus lactis [ 19 ]. In addition, dr_1558 enhances the production of succinic acid [ 22 ], γ-aminobutyric acid (GABA) in E. coli [ 23 ], and cadaverine in Corynebacterium glutamicum [ 24 ]. Moreover, the biosynthesis of poly(3-hydroxybutyrate) (PHB), a biopolymer that can replace petroleum-based plastics [ 25 ], was highly produced in synthetic E. coli strains under dr_1558 regulation. To date, 67 Deinococcus- type strains have been isolated and sequenced. However, most studies have focused on the D. radiodurans genes. Recently, Lim et al. reported that complicated stress resistance systems were widely located in the genome of Deinococcus as conserved and divergent forms [ 26 ], which are not fully understood yet. In this study, to confer oxidative stress resistance and cellular robustness on E. coli , we explored the genomic library of Deinococcus proteolyticus. Through a series of screening steps, we selected a gene that has the potential to resist a high concentration of hydrogen peroxide. The selected gene was introduced subsequently to the PHB-generative E. coli strain to investigate its impact on PHB synthesis and metabolism.",
"discussion": "4. Discussion In this study, we found that D. proteolyticus exhibited high resistance potential toward oxidative stress. In the core-genome analysis between D. proteolyticus and D. radiodurans , important genes for stress resistance were observed in both strains with high similarity, which was also observed in the comparative analysis of 11 genomes in the Deinococcus species [ 26 ]. TCSs facilitate the detection of environmental signals and regulate diverse stress-resistance-associated genes [ 37 ]. Among the stress response genes in D. proteolyticus , we revealed that D. proteolyticus has 11 two-component systems, 6 orphan histidine kinases, and 6 orphan response regulators. A comparative analysis with the genome of D. radiodurans showed that most of the response regulators (73%) have a high degree of similarity (>50%) with response regulators from D. radiodurans ( Supplementary Figure S3 ). It was proposed that highly preserved response regulators may play a pivotal role in the stress response. From a genomic library constructed using the gDNA of D. proteolyticus , we isolated a gene (Deipr_0871) that enhanced the oxidative stress resistance of E. coli strains. The selected clone contained a response regulator (RR) domain involved in a two-component signal transduction system (TCSs) with histidine kinase (HK) [ 37 ]. Deipr_0871 was composed of two domains, receiver domain and NarL-like LuxR type-HTH domain. The NarL/FixJ family has divergent roles in bacterial systems such as nitrogen fixation and sugar phosphate transport [ 38 ]. A phylogenetic tree with other known NarL family regulators revealed that Deipr_0871 was found in close proximity to C. salexigens EupR, E. coli NarP, and NarL response regulators ( Figure 3 ). C. salexigens EupR was previously recognized as playing a function in compatible solute absorption, whereas NarP and NarL can regulate nitrogen metabolism. The heterologous expression of DR_1558, a NarL-like family response regulator in D. radiodurans, resulted in similar phenotypic properties with those of deipr_0871, such as high oxidative stress resistance, despite their low homology (38%). A previous study revealed that the oxidative stress resistance of the E. coli strain harboring DR_1558 was enhanced via an increase in rpoS transcripts. Additionally, other DR_1558 homologs in Antarctic bacteria can upregulate rpoS transcripts [ 13 ]. RpoS is an alternative sigma factor that plays a central role in adaptation to many suboptimal growth conditions by controlling the expression of many genes, including those affecting phenotypic traits such as metabolic pathways and the expression of genes required to survive nutrient deprivation [ 20 ]. Multiple alignments of amino acid composition located in the putative DNA binding site suggested that Deipr_0871 may control the stress resistance of E. coli strains in a manner different from that of other regulators. It was also confirmed by a transcriptional change in the recombinant E. coli strain in the presence of deipr_0871. The heterologous expression of deipr_0871 did not regulate rpoS or its downstream genes ( Figure 4 B). Under normal growth conditions, SodA and SoxS transcripts were highly expressed in the presence of deipr_0871 , which increased the stress resistance of the E. coli strain. In E. coli , two major regulatory defense systems respond to oxidative stress: oxy R and sox RS. [ 36 ]. OxyR responds to hydrogen peroxide and induces the expression of catalases such as kat G, dps , and ahp C, whereas SoxRS responds to redox-active compounds and regulates superoxide dismutase (SodA). [ 36 ]. SodA plays a role in decreasing the levels of cytotoxic ROS [ 39 ], which has the properties of cellular responses to oxidative stresses for detoxification [ 36 ]. Moreover, SoxS can improve NADPH pools and promote antioxidant defense by mediating the reduction of thioredoxins or glutaredoxins [ 40 , 41 ]. According to Shery et al. and Henard et al., SoxS activates the expression of genes involved in carbon metabolic pathways, such as glycolysis and the TCA cycle [ 40 , 41 ]. The SoxS deletion mutant shows reduced glucose uptake and growth rates [ 42 ]. This can explain the effect of deipr_0871 on growth, as the growth rate in the presence of deipr_0871 was higher than that in the control strain. Interestingly, glucose supplementation altered the metabolic ability of the E. coli strain in the presence of deipr_0871 , which boosts the growth rate and cell density of the E. coli ( Figure 6 ). In addition, it stimulated PHB production in the recombinant E. coli strain. PHB is a bioplastic produced intracellularly by microorganisms to save energy as a carbon source [ 43 , 44 ]. The sufficient provision of acetyl-CoA and the availability of the cofactor NADPH significantly impact the synthetic efficiency of PHB, and metabolic engineering to expand the supply of NADPH has been attempted to boost PHB production [ 45 ]. As shown in Figure 7 , this suggests that Deipr_0871 differentially regulates the central carbon metabolism compared to DR_1558. NADPH-generating genes ( zwf, icdA, sucA, and mdh ) in the pentose phosphate pathway and TCA cycle were upregulated in the presence of deipr_0871, indicating an improvement in cell growth by generating energy (ATP, NADPH, etc.) through the pentose phosphate and TCA pathways. The higher expression levels of genes related to the pentose phosphate pathway, glycolysis, and TCA cycle produce more energy in the form of ATP, NADH, and NADPH for cell growth, resulting in the increased proliferation period of pRad-0871 strains. High concentrations of NADPH also activate acetoacetyl-CoA reductase ( phaB ) involved in the synthesis of PHB. In addition, the overflow of glycolysis and the TCA pathway generate and accumulate numerous pyruvate and acetyl-CoA precursors of the product (PHB), which are elevated. Using a genomic library of D. proteolyticus , we isolated the NarL-like response regulator Deipr_0871. The introduction of deipr_0871 in E. coli showed improved oxidative stress resistance and growth rate through the soxS regulation system. This is different from the other Deinococcal NarL-like response regulator, DR_1558. Moreover, its mechanism in metabolic pathways and stress resistance was employed to improve PHB production by boosting the acetyl-CoA and NADPH generation pathways, respectively. It concluded that deipr _ 0871 can also be employed to improve the capabilities of industrial strains."
} | 2,960 |
35236782 | PMC8906998 | pmc | 6,164 | {
"abstract": "Neurons-on-a-Chip technology has been developed to provide diverse in vitro neuro-tools to study neuritogenesis, synaptogensis, axon guidance, and network dynamics. The two core enabling technologies are soft-lithography and microelectrode array technology. Soft lithography technology made it possible to fabricate microstamps and microfluidic channel devices with a simple replica molding method in a biological laboratory and innovatively reduced the turn-around time from assay design to chip fabrication, facilitating various experimental designs. To control nerve cell behaviors at the single cell level via chemical cues, surface biofunctionalization methods and micropatterning techniques were developed. Microelectrode chip technology, which provides a functional readout by measuring the electrophysiological signals from individual neurons, has become a popular platform to investigate neural information processing in networks. Due to these key advances, it is possible to study the relationship between the network structure and functions, and they have opened a new era of neurobiology and will become standard tools in the near future.",
"introduction": "INTRODUCTION The brain is a large complex network. One of the key questions in neuroscience research has been how the complicated functions of the brain emerge from the intrinsic structures of the neural networks. To understand the underlying mechanism of the brain, dissociated neuronal cultures have been widely used as an in vitro model. Neurons, grown on conventional cell culture dishes, can extend processes, dendrites and axons for target selection and express ion channels for action potential generation. Moreover, they can form functional synapses between themselves, making it possible to investigate not only individual cells but also neuronal networks. Above all, in vitro networks have the advantages of better accessibility and easier manipulation compared with in vivo conditions ( Feldt et al., 2011 ). To establish neuronal networks in vitro , neurons from dissociated tissues adhere onto a culture substrate and their processes begin to grow and connect with each other. At this time, however, the positions and connections are randomly determined. This randomness makes the organized networks of dissociated neurons vastly different from the well-ordered neural networks in the brain and leads to a lack of reproducibility as an experimental model. To deal with this limitation, various cell patterning methods have been developed based on microfabrication technologies. These techniques have enabled the control of the network structures and complemented the random organization. It has also facilitated a study on the relationship between structure and function by measuring and analyzing the electrophysiological activity of the network whose structure formed to have a desired design ( Hasan and Berdichevsky, 2016 )."
} | 725 |
28757971 | PMC5508654 | pmc | 6,165 | {
"abstract": "A new solar-microbial hybrid device based on oxygen-deficient Nb 2 O 5 anodes for sustainable hydrogen generation without external bias was demonstrated.",
"conclusion": "4. Conclusions In summary, we developed a facile and effective method to significantly improve the conductivity and performances of the Nb 2 O 5– x NPs films in PEC cells and MFCs through the introduction of oxygen vacancies. The Nb 2 O 5– x NPs photoanode achieved a remarkable photocurrent density of 0.9 mA cm –2 at 0.6 V ( vs. SCE) in a 1 M KOH aqueous solution. Meanwhile, the Nb 2 O 5– x -MFC exhibited a superior power density of 1196 mW m –2 when the Nb 2 O 5– x NPs film was used as an anode in the MFC cell. The oxygen vacancy plays a critical role in enhancing the effective charge transport of the electrodes, as well as increasing the conductivity. Moreover, we also demonstrated that the PEC–MFC hybrid device with the Nb 2 O 5– x NPs anodes in both devices is feasible and could produce hydrogen even at zero external bias (0 V vs. Pt) under illumination. To our best knowledge, it is the first report about the applications of Nb 2 O 5 materials in an integrated PEC–MFC device to produce hydrogen without an external bias, by just using organic matter and solar light. This environmentally friendly and novel design provides a promising research direction for the future development of energy conversion and storage.",
"introduction": "1. Introduction The rational utilization of renewable and clean energy has drawn more and more attention due to the increasing energy demands and ever-growing environmental concerns. Producing hydrogen by photoelectrochemical (PEC) water splitting over a semiconductor photoelectrode is emerging as the most promising way that can directly convert solar energy into clean hydrogen. 1 – 3 Hydrogen from water splitting is known to be a thermodynamically uphill or endothermic process and can be described as the following equation: 1 H 2 O(l) → H 2 (g) + 1/2O 2 (g), E 0 = 1.23 V vs. NHE \n To drive this reaction, a minimal energy of 237.2 kJ mol –1 that is equal to a potential of 1.23 V vs. NHE is required. 2 , 3 In this respect, it requires the conduction band (CB) and valence band (VB) edges of the photoelectrode to straddle the reduction and oxidation potentials of water. Specifically, E \n CB should be above E \n red (H 2 /H + ) and E \n VB should be below E \n ox (OH – /O 2 ). Besides having suitable band edge positions, an ideal photoelectrode material for efficient solar water splitting should also possess strong light-harvesting ability, good chemical stability, fast charge transport and low cost. 4 – 6 However, most of the developed photoelectrodes cannot satisfy these requirements simultaneously. On the other hand, an external bias is always needed for practical applications to improve their charge separation and/or overcome an overpotential for the low lying CB edge, which leads to increased cost of hydrogen production. 7 – 10 In order to address the above-mentioned energy loss, numerous efforts have been devoted to designing nanostructured photoelectrodes and catalysts as well as optimizing the PEC configuration. 11 – 13 Nevertheless, the current efficiency of PEC water splitting is still not satisfactory for its high energy demand. 14 , 15 Alternatively, the energy required for water splitting obtained from a renewable energy source is a more cost-effective approach to generate hydrogen. Microbial fuel cells (MFCs) are of great interest since they can directly produce electricity from organic waste and biomass at a low cost. MFCs are bioelectrochemical devices that can convert chemical energy stored in organic matter into electricity with the help of microorganisms. 16 – 23 This feature makes them very attractive as a green energy supply to overcome the thermodynamic constraints and compensate for the energy loss of PEC water splitting. Recently, Wang et al. demonstrated that the hydrogen gas could be continuously produced based on solar light and biomass recycling through coupling PEC water splitting with a TiO 2 nanowire-arrayed photoanode and microbial electrohydrogenesis with Shewanella oneidensis MR-1 in a PEC–MFC hybrid device. 16 Despite these achievements, the unsatisfactory performances of the current MFCs and PEC cells are the main obstacles for their practical applications. The electrode material is the most key factor that determines the performances of both the MFCs and PEC cells. 16 – 19 , 24 – 27 Therefore, the exploration and development of an advanced electrode material for improving the efficiency of PEC cells and the output power density of the MFCs are highly desirable. In this work, we demonstrate the feasibility of oxygen-deficient Nb 2 O 5 nanoporous (Nb 2 O 5– x NPs) films as a high-performance anode material for both the PEC cells and MFCs. Nb 2 O 5 is one of the most important n-type semiconductor materials for dye-sensitized solar cells and photocatalysts in terms of its excellent photoactivity, non-toxicity and environmental friendliness. 28 – 33 It has a similar bandgap to TiO 2 and ZnO, and favourable band-edge positions that can straddle the redox potential of water photoelectrolysis. Moreover, recent reports have shown that Nb 2 O 5 possesses better biocompatibility than TiO 2 and higher stability than ZnO (amphoteric oxide). 34 , 35 All of these characteristic features make it a very promising photoanode and anode for PEC cells and MFCs, respectively. However, Nb 2 O 5 suffers from poor electrical conductivity, which seriously limits its wide applications. Herein, we developed a facile approach to significantly boost the conductivity of Nb 2 O 5 by creating oxygen vacancies. Nb 2 O 5– x NPs films were readily obtained by anodic oxidation of Nb foil and hydrogenation treatment. Benefiting from the appropriate bandgap, suitable band levels, improved conductivity and good biocompatibility, the Nb 2 O 5– x NPs films exhibited superior performances in both the PEC cells and MFCs. The Nb 2 O 5– x photoanode achieved a remarkable photocurrent density of 0.9 mA cm –2 at 0.6 V ( vs. SCE) in 1 M KOH aqueous solution, and the MFCs with the Nb 2 O 5– x NPs anodes (denoted as Nb 2 O 5– x -MFCs) exhibited a maximum power density of 1196 mW m –2 . More interestingly, a PEC–MFC hybrid device, by interfacing a Nb 2 O 5– x -based PEC device and a Nb 2 O 5– x -based MFC device, was designed and continuous hydrogen gas could be produced at zero external bias by biodegradable organic matter and solar light.",
"discussion": "3. Results and discussion 3.1 Synthesis and characterization of Nb 2 O 5– x NPs films Nb 2 O 5– x NPs films were obtained through a two-step procedure, which involves the anodic oxidation of Nb foil and heat treatment in a hydrogen atmosphere. Firstly, the Nb 2 O 5 NPs films were synthesized on a niobium film substrate by a simple anodic oxidation method and annealed in air at 450 °C (Experimental section). The SEM images show that the white homogeneous film obtained on the metal substrate is composed of dense nanoporous arrays with ∼50 nm diameter (Fig. S1, ESI † ). To introduce oxygen vacancies into the Nb 2 O 5 NPs films, the as-prepared Nb 2 O 5 NPs films were then annealed in a hydrogen atmosphere for an additional 60 min at 500 °C. The synthetic process of the hydrogen treatment is illustrated in Fig. 1a . After the treatment, the film colour changed from white to blue (Fig. S2, ESI † ), suggesting a possible modification in the crystal structure. To identify the possible phase transformation, XRD patterns of the Nb 2 O 5 and Nb 2 O 5– x NPs films are shown in Fig. 1b . Sharp diffraction peaks centered at 2 θ angles of 22.6°, 28.4° and 50.8°, corresponding to the (001), (180) and (0 16 0) planes of Nb 2 O 5 (JCPDS #30-0873), are observed for both samples, indicating that the NPs films are well crystalline with a similar phase. SEM studies also reveal there are no obvious morphological changes for the Nb 2 O 5– x NPs film after the hydrogenation ( Fig. 1c ). Fig. 1d displays the TEM image and the selected-area electron diffraction (SAED) of the Nb 2 O 5– x NPs film. The TEM image obviously shows that the porous Nb 2 O 5– x sample consists of the nanoporous arrays. The clear lattice fringes with a spacing of 0.39 nm are indexed to the (001) plane of the orthorhombic Nb 2 O 5 samples. Meanwhile, the bright and well-arranged diffraction spots further confirm the highly crystalline nature of the Nb 2 O 5– x NPs film. Fig. 1 (a) Crystal structure of Nb 2 O 5 and showing the formation of oxygen vacancies; (b) XRD spectra of Nb 2 O 5– x NPs film; (c) SEM images of Nb 2 O 5– x NPs film; inset: magnified SEM image of Nb 2 O 5– x NPs film; (d) TEM images of Nb 2 O 5– x NPs film; the upper left inset: lattice-resolved TEM image collected at the edge of the NP film; the bottom right inset: the diffraction pattern; (e) normalized Nb 3d core-level XPS spectra collected for Nb 2 O 5 and Nb 2 O 5– x NPs films. The UV-visible absorption spectra of Nb 2 O 5 and Nb 2 O 5– x NPs films were collected to investigate the influence of hydrogenation on the optical absorption (Fig. S2, ESI † ). Both of the samples exhibit similar light absorption in the UV region and the Nb 2 O 5– x NPs film shows substantially higher absorption when compared to pristine Nb 2 O 5 in the region of 400–800 nm, which suggests Nb 2 O 5– x may absorb more visible light. The band gap of the Nb 2 O 5– x NPs film is calculated to be 3.26 eV, while it is 3.35 eV for the pristine Nb 2 O 5 NPs film, indicating the hydrogenation has a negligible effect on its band gap. To further investigate the effect of the hydrogen treatment on the chemical state of Nb 2 O 5 , XPS analysis was performed. The XPS survey of the Nb 2 O 5– x NPs film was collected to prove that no other impurities were introduced after the hydrogen treatment (Fig. S3, ESI † ). Fig. 1e displays the normalized high resolution Nb 3d core level XPS spectra. Both of the samples have two peaks located at 207.3 and 210 eV, which are ascribed to the regular Nb 3d signals for Nb 5+ . 36 , 37 Remarkably, the Nb 2 O 5– x NPs film displays an additional peak which emerged at the lower binding energy of 205.9 eV, which is the typical peak position for the low charge Nb 4+ . 38 – 40 The presence of Nb 4+ in the Nb 2 O 5– x NPs film also had been regarded as the reason why the film’s colour changed from white to blue after the hydrogenation (Fig. S2, ESI † ). 41 – 43 \n Fig. 2a compares the normalized O 1s core level XPS spectra of the Nb 2 O 5 and Nb 2 O 5– x NPs samples. Significantly, the Nb 2 O 5– x NPs film shows a broader peak at around 531.68 eV than that of the Nb 2 O 5 NPs film, indicating it has more oxygen vacancies than the Nb 2 O 5 NPs film. 40 , 44 – 48 This is also confirmed by the electron paramagnetic resonance (EPR) analysis. The broader peaks at g = 2.15 and 2.02 in Fig. 2b clearly reveal the density of oxygen vacancies in the Nb 2 O 5– x NPs film is much higher than that in the Nb 2 O 5 NPs film. 49 , 50 \n Fig. 2 (a) Normalized O 1s core-level XPS and (b) EPR spectra collected for Nb 2 O 5 and Nb 2 O 5– x NPs films. Electrochemical impedance measurements were carried out to verify our hypothesis that the induced oxygen vacancy can serve as a shallow donor to increase the carrier density of Nb 2 O 5 . Fig. 3a displays the Mott–Schottky plots of the electrodes at a frequency of 1 kHz in the dark, which are generated based on capacitances that derived from the electrochemical impedance. Both the Nb 2 O 5 and Nb 2 O 5– x NPs films show positive slopes, in line with the characteristic of a n-type semiconductor. Notably, the Nb 2 O 5– x NPs film shows a substantially smaller slope for the Mott–Schottky plot than the Nb 2 O 5 NPs film, suggesting a significantly increased donor density, based on the following equation: 3 N d = (2/ e 0 εε 0 )[d(1/ C 2 )/d V ] –1 where N \n d is the donor density, e \n 0 is the electron charge, ε is the dielectric constant of Nb 2 O 5 ( ε = 41), ε \n 0 is the permittivity of vacuum, and V is the applied bias at the electrode. 41 The carrier densities of the Nb 2 O 5 and Nb 2 O 5– x samples are calculated to be 1.45 × 10 18 and 3.68 × 10 23 cm –3 , respectively. It should be noted that here we used the electrode area instead of the surface area of the nanoporous film for the calculation, which may cause errors in determining the carrier densities. However, a qualitative comparison of the carrier densities between the Nb 2 O 5 and Nb 2 O 5– x NP samples is reliable, since they have a similar morphology and surface area. Obviously, the Nb 2 O 5– x NPs film possesses a 5 order of magnitude improvement on the donor density compared to the Nb 2 O 5 NPs film, which results in a significant facilitation of the charge separation in the Nb 2 O 5– x NPs film. Meanwhile, the I – V curves of the Nb 2 O 5 and Nb 2 O 5– x NPs samples at room temperature were also collected in Fig. 3b . As expected, the electrical conductivity of the Nb 2 O 5 sample is substantially improved after hydrogenation. All of these results validly confirm that the introduced oxygen vacancies can remarkably enhance the donor density as well as the conductivity of the Nb 2 O 5 NPs film. Fig. 3 (a) Mott–Schottky plots of Nb 2 O 5 and Nb 2 O 5– x NPs films at a frequency of 1 kHz in the dark; (b) comparison of I – V curves of the samples; the inset is the schematic of the measurement. 3.2 Performances of PEC devices based on Nb 2 O 5– x photoanodes The PEC performances of the Nb 2 O 5 samples were measured in a three electrode electrochemical cell with a 1.0 M NaOH solution as the electrolyte. For optimizing the PEC performance of the Nb 2 O 5– x NPs film, the effect of the hydrogenation temperature was studied. Fig. 4a shows the linear sweep curves (LSV) of the pristine Nb 2 O 5 and Nb 2 O 5– x NPs films obtained at different hydrogenation temperatures. The photocurrent density of the Nb 2 O 5 NPs film is 0.05 mA cm –2 at 0.6 V vs. SCE, which is close to the value reported previously. 36 The photocurrent densities of the Nb 2 O 5– x NPs films increase gradually with the increase of the hydrogen treatment temperature from 450 to 500 °C. The Nb 2 O 5– x (500 °C) NPs film achieves a maximum value of 0.9 mA cm –2 at 0.6 V vs. SCE, which is about a 20-fold enhancement compared to the Nb 2 O 5 NPs film at the same potential. This photocurrent density is also dramatically higher than the values recently reported for the reported values of Nb 2 O 5 photoanodes, such as N-doped Nb 2 O 5 nanostructures (Table S1 † ). 36 , 51 – 53 Photocurrent densities decrease gradually with the rising temperature when the annealing temperature is above 500 °C. According to the increasing donor density and conductivity of the Nb 2 O 5– x with the rising hydrogenation temperature (Fig. S4, ESI † ), it can be concluded that excessive oxygen vacancies will serve as recombination centres for electrons and holes, resulting in a poor PEC performance, in agreement with other work. 54 To quantitate the interplay between the photoactivity and light absorption, incident photon to current conversion efficiencies (IPCE) were measured for the Nb 2 O 5 and Nb 2 O 5– x NPs photoanodes at 0.2 V vs. SCE ( Fig. 4b ). All of the Nb 2 O 5– x NPs films show significantly enhanced photoactivity over the entire UV region, and the Nb 2 O 5– x NPs film hydrogenated at 500 °C exhibits the best IPCE efficiency in the wavelength range from 300 to 380 nm. Additionally, the IPCE values decrease from 35% at 300 nm to 1% at 400 nm, and we did not observe any photoactivity in the visible light region beyond 400 nm, indicating that the observed colour change is not because of the band gap modification of Nb 2 O 5 or the transition between the impurity states and conduction/valence band edges. 47 Thus, we believe that the photoactivity enhancement of the Nb 2 O 5– x NPs film is because of the increased donor density and conductivity originating from the induced oxygen vacancies generated during the hydrogenation. Fig. 4 (a) I – V curves measured under 100 mW cm –2 simulated solar light generated by a 100 W xenon lamp coupled with an AM 1.5G filter in 1 M NaOH electrolyte; (b) IPCE spectra measured at 0.2 V vs. SCE in 1 M NaOH electrolyte. 3.3 Performances of the MFC devices based on the Nb 2 O 5– x anodes To demonstrate that the as-prepared Nb 2 O 5– x NPs film is also a promising candidate as a high-performance anode material for a MFC device, a simple MFC device was assembled using the Nb 2 O 5– x NPs film hydrogenated at 500 °C as the anode and a 40 wt% Pt/C loaded carbon paper as the air cathode in the E. coli MFCs (denoted as Nb 2 O 5– x -MFC, Experimental section). For a better comparison, the performance of the pristine Nb 2 O 5 NPs film in the MFC device was also measured (denoted as Nb 2 O 5 -MFC). Fig. 5a and b depict the comparison of the polarization curves and power outputs tested by loading various external resistances. In comparison to the Nb 2 O 5 -MFC, the cell voltage of the Nb 2 O 5– x -MFC decreased more slowly with the decreased loaded resistance and gently, indicating the superior performance of the Nb 2 O 5– x NPs anode. Furthermore, the Nb 2 O 5– x -MFC achieved a remarkable maximum power density of 1196 mW m –2 at a current density of 4465 mA m –2 , which is substantially higher than that of the Nb 2 O 5 -MFC (140 mW m –2 ). The distinct enhancement between the Nb 2 O 5 and Nb 2 O 5– x MFC devices can be ascribed to the superior conductivity of the Nb 2 O 5– x NPs anode. Additionally, without E. coli , the Nb 2 O 5– x -MFC device shows negligible cell voltage and output power density when loading the external resistances (Fig. S5 † ), indicating E. coli is very important for power production. Fig. 5 Comparisons of (a) polarization curves and (b) power outputs for Nb 2 O 5 and Nb 2 O 5– x anodes. 3.4 Performances of PEC–MFC hybrid devices based on Nb 2 O 5– x anodes For taking full advantage of the oxygen-deficient electrodes, we assembled a PEC–MFC hybrid device to convert and store energy using the Nb 2 O 5– x NPs film as the anode in both the MFC and the PEC cell (Experimental section). The PEC properties of Nb 2 O 5– x NPs film were measured in a two-electrode electrochemical cell with/without the MFC devices in the dark and under 1 sun illumination. Fig. 6a shows the schematic configuration of the integrated PEC–MFC hybrid device. By coupling the PEC and MFC devices in series, the Nb 2 O 5– x anode in the MFC provides a biovoltage that shifted the potential of the illuminated Nb 2 O 5– x photoanode near to –0.6 V, thus, enabling water splitting to occur at zero external bias ( Fig. 6b ). The PEC–MFC device displays a novel photocurrent density of 0.18 mA cm –2 at zero bias (0 V vs. Pt), which is substantially larger than the one obtained from the PEC cell alone at the same potential ( Fig. 6b ). More importantly, gas bubbles were clearly observed to be continuously evolving on the Pt electrode under light illumination (Fig. S6 and Video S1, ESI † ), indicating the generation of hydrogen. Meanwhile, the PEC device in the presence of glucose, possessing a lower performance than the hybrid device, was also conducted to emphasize the excellent energy efficiency via this PEC–MFC hybrid device (Fig. S7 † ). There is no doubt that the Nb 2 O 5– x NPs film is an outstanding material and its merits can be integrated into both a PEC cell, MFC and a hybrid device and this hybrid device is an efficient strategy to convert and store energy. The PEC–MFC device also exhibits reproducible photocurrent generation in response to light illumination ( Fig. 6c ), implying the hybrid device is feasible to produce hydrogen. To the best of our knowledge, this is the first demonstration of using PEC–MFC hybrid devices based on the same anode material to generate hydrogen at zero external bias. Fig. 6 (a) Schematic configuration of a PEC–MFC device; (b) I – V curves collected from a PEC device (red line) and a PEC–MFC device (olive line) with the Nb 2 O 5– x NPs electrodes at a scan rate of 10 mV s –1 with/without the white light illumination; (c) I – T curves recorded for the PEC device (red line) and the PEC–MFC device (olive line) at 0 V vs. Pt, with light on/off cycles."
} | 5,126 |
22355664 | PMC3216628 | pmc | 6,166 | {
"abstract": "Under high-strain-rate compression (strain rate ∼10 3 s −1 ), nacre (mother-of-pearl) exhibits surprisingly high fracture strength vis-à-vis under quasi-static loading (strain rate 10 −3 s −1 ). Nevertheless, the underlying mechanism responsible for such sharply different behaviors in these two loading modes remains completely unknown. Here we report a new deformation mechanism, adopted by nacre, the best-ever natural armor material, to protect itself against predatory penetrating impacts. It involves the emission of partial dislocations and the onset of deformation twinning that operate in a well-concerted manner to contribute to the increased high-strain-rate fracture strength of nacre. Our findings unveil that Mother Nature delicately uses an ingenious strain-rate-dependent stiffening mechanism with a purpose to fight against foreign attacks. These findings should serve as critical design guidelines for developing engineered body armor materials.",
"discussion": "Discussion Quantitatively, the activation volume (v*) of plastic deformation, delineating the physical domain of plastic deformation events, can be linked to the strain rate sensitivity (SRS) 27 as Here k is the Boltzmann constant (1.38×10 −23 m 2 kg s −2 k −1 ), T is absolute temperature, and σ and m are respectively the effective stress and the SRS. Clearly the activation volume can be used as a finger-print of the deformation mechanisms. Based on the experimental results of this work, the SRS of nacre is in the order of ∼0.1, which in turn corresponds to an activation volume in the order of a few b 3 (Section 2 in supplementary materials). This implies that local plastic deformation is readily activated upon dynamic loading, even though the plasticity may not be sustainable as evidenced from the lack of global plastic deformation of the dynamic specimens. As a result, relatively “harder” physical mechanisms, i.e., partial dislocation emission and deformation twinning (see more deformation twinning in section 3 of supplementary materials), debut as increasing applied stress induced by enhancing strain rate. In contrast, comparatively “easier” dislocation mechanisms, such as dislocation slip and grain boundary rearrangement, are prevalent owing to large activation volumes at lower loading rates 27 . We believe that the emerging partial dislocations and deformation twinning in nacre are strongly correlative with the miniaturized activation volume. Finally, a significant fact should be noted that on the basis of our evaluation (Section 4 in supplementary materials) individual aragonite platelets act as dominating “carriers” in overall strength enhancement, whereas the contribution of biopolymer to rate-strengthening remains quite limited due to its low volume fraction (5 vol. %) as well as its low mechanical stiffness. In orthorhombic aragonite, the atomic configuration and crystal structure are far more complicated than those more common FCC/BCC metals. We suggest that dynamic loading and the extremely small dimensions of the aragonite nanoparticles work together to restrain full dislocation-related activities. As a consequence, nacre appeals to the alternative harder mechanisms such as partial dislocations and deformation twinning, explaining its dynamic self-stiffening behavior. These findings significantly advance our understanding of nacre's design principles and provide guidelines for optimizing the damage tolerance design of engineered nacre-inspired materials. We have to note, though, that the formation and evolution mechanisms of partial dislocations and deformation twinning in orthorhombic aragonite still remain an open question to be explored. Atomistic simulation and modeling may help to shed light into this arena. Another question that may follow is: What are the structural details at the nanoparticle boundary/interface in nacre after dynamic loading? Our previous TEM results 15 showed that at least two types of imperfect transition exist in pristine nacre: screw dislocation and amorphous aggregation. A large number of dislocation events from particle boundary/interface are believed to result in an irregular and complex atomic configuration. Figure 5a demonstrates an amorphous aggregation regime encircled (white boxed area) by nanoparticles after dynamic compression, where the messy spot diffraction pattern (inset) indicates that previously ordered arrangements of aragonite nanoparticles in pristine nacre have been completely destroyed. The amorphous phase is further confirmed by FFT ( Figure 5b ) that scatters into a halo feature. An extremely important detail is that the amorphous zone extends to at least four nanometers in size ( Figure 5c ), which was not observed in the fresh and the quasi-statically deformed nacre samples. The direct response is the elongation of the pre-existing amorphous particle boundary (two nanometers in size at most, based on our observation). However, although no direct evidence is at hand, we suggest that the metastable aragonite crystalline phase has probably in part transformed to an amorphous phase, which is extensively found in the dynamically compressed nacre samples along c-axis. A similar case was reported by Chen and co-workers in ballistically tested boron carbide 28 . We expect that such phase transformation may improve nacre's high-strain rate performance. In addition, our observation conversely confirms the previous conclusion 15 that amorphous aggregation is an important transition phase acting as “grain boundaries” between the nanoparticles. In summary, we demonstrate for the first time solid evidence that two new deformation mechanisms, partial dislocation emission and deformation twinning, are triggered in nacre's aragonite platelets under high-strain-rate uniaxial compression, which renders a much elevated fracture strength in comparison to quasi-static loading. This dynamic self-stiffening behavior benefits nacre's self-protection from predatory penetrating attack. These findings advance our understanding of damage tolerance design principle in engineered body armor materials."
} | 1,526 |
27066229 | PMC4782245 | pmc | 6,168 | {
"abstract": "Abstract Changes in soil nutrient availability during long‐term ecosystem development influence the relative abundances of plant species with different nutrient‐acquisition strategies. These changes in strategies are observed at the community level, but whether they also occur within individual species remains unknown. Plant species forming multiple root symbioses with arbuscular mycorrhizal ( AM ) fungi, ectomycorrhizal ( ECM ) fungi, and nitrogen‐(N) fixing microorganisms provide valuable model systems to examine edaphic controls on symbioses related to nutrient acquisition, while simultaneously controlling for plant host identity. We grew two co‐occurring species, Acacia rostellifera (N 2 ‐fixing and dual AM and ECM symbioses) and Melaleuca systena ( AM and ECM dual symbioses), in three soils of contrasting ages ( c . 0.1, 1, and 120 ka) collected along a long‐term dune chronosequence in southwestern Australia. The soils differ in the type and strength of nutrient limitation, with primary productivity being limited by N (0.1 ka), co‐limited by N and phosphorus (P) (1 ka), and by P (120 ka). We hypothesized that (i) within‐species root colonization shifts from AM to ECM with increasing soil age, and that (ii) nodulation declines with increasing soil age, reflecting the shift from N to P limitation along the chronosequence. In both species, we observed a shift from AM to ECM root colonization with increasing soil age. In addition, nodulation in A. rostellifera declined with increasing soil age, consistent with a shift from N to P limitation. Shifts from AM to ECM root colonization reflect strengthening P limitation and an increasing proportion of total soil P in organic forms in older soils. This might occur because ECM fungi can access organic P via extracellular phosphatases, while AM fungi do not use organic P. Our results show that plants can shift their resource allocation to different root symbionts depending on nutrient availability during ecosystem development.",
"introduction": "Introduction Many terrestrial plants form symbiotic associations with soil biota to enhance nutrient acquisition. The most widespread of these associations involves mycorrhizal fungi (Fig. 1 ), which occur in roots of >80% of all plant species (Wang and Qiu 2006 ; Brundrett 2009 ). The two main types of mycorrhizas are arbuscular mycorrhizas (AM) and ectomycorrhizas (ECM). Arbuscular mycorrhizas enhance the acquisition of inorganic phosphorus (P) and other relatively immobile nutrients, while ectomycorrhizas also allow plants to access both organic nitrogen (N) and P, as well as sorbed P (Hodge et al. 2001 ; Leigh et al. 2009 ; Plassard and Dell 2010 ). Some plant species also form root symbiotic associations with N 2 ‐fixing bacteria in nodules, allowing plants to acquire atmospheric N (Gutschick 1984 ). Figure 1 Cleared and stained roots showing arbuscular mycorrhizas (right panel) and ectomycorrhizas (left panel). Plants allocate substantial amounts of carbon (C) to sustain symbiotic associations with mycorrhizal fungi or N 2 ‐fixing bacteria (Pate and Herridge 1978 ; Smith and Read 2008 ). Carbon allocation to AM and ECM fungi can represent >20% of the total C fixed daily in photosynthesis (Bryla and Eissenstat 2005 ; Hobbie 2006 ). Likewise, C allocation to nodules by N 2 ‐fixing plant species can represent >30% of daily photosynthates (Minchin and Pate 1973 ). However, plant investment in symbiotic associations depends strongly on plant nutrient requirements and soil nutrient availability (van der Heijden 2001 ; Lambers et al. 2008 ). The occurrence of AM fungi tends to be more common in neutral soils with low P availability and low organic matter content (Johnson et al. 1991 ; Coughlan et al. 2000 ; Smith et al. 2015 ). By contrast, ECM fungi are more common in acidic soils with lower mineral N concentrations and higher organic matter content (van der Heijden and Kuyper 2001 ; Lilleskov et al. 2002 ). Nitrogen fixation plays a greater role in N acquisition at low soil N availability and is inhibited by N fertilization (Imsande 1986 ; Kanayama et al. 1990 ). These studies suggest that plants decrease investment in root symbionts when nutrient supply is high, thus allocating C in a manner that increases acquisition of the nutrients that most strongly limit their growth. Most plant species form associations with only one type of mycorrhizal fungi (e.g., AM or ECM). However, some plant species form dual associations with both AM and ECM fungi (Cázares and Smith 1996 ; Chen et al. 2000 ; Adams et al. 2006 ; Pagano and Scotti 2008 ), and, in some cases, a tripartite root symbiosis involves N 2 ‐fixing microorganisms (e.g., Acacia holosericea ; Founoune et al. 2002 ). Several studies have shown negative correlations between AM and ECM fungi, and this relationship may reflect competitive exclusion of AM fungi by ECM fungi (Lapeyrie and Chilvers 1985 ; Lodge and Wentworth 1990 ; Neville et al. 2002 ). On the other hand, positive relationships between nodulation and both AM and ECM colonization have been reported (Founoune et al. 2002 ; André et al. 2003 ; Lesueur and Duponnois 2005 ). The reliance of plants on root symbionts can be better understood by studying within‐species shifts in root symbionts with changing soil properties and plant N:P stoichiometry (Jones et al. 1998 ; Founoune et al. 2002 ; Neville et al. 2002 ). Such shifts have rarely been studied (but see Neville et al. 2002 ; Nilsson et al. 2005 ); hence, further research needs to identify factors involved in the balance between multiple symbioses. Long‐term soil chronosequences (i.e., gradients of soil age) offer valuable “natural experiments” to study how soil nutrient availability and stoichiometry influence plant–soil interactions (Walker et al. 2010 ; Turner and Condron 2013 ). During tens to hundreds of thousands of years of soil and ecosystem development, changes in soil and plant communities co‐occur that strongly alter soil nutrient dynamics (Walker and Syers 1976 ; Wardle et al. 2004 ; Peltzer et al. 2010 ). In young soils, pH is higher, P is most abundant, and N is generally the key limiting nutrient (Walker and Syers 1976 ; Turner and Laliberté 2015 ). As soils develop, pH decreases, soil N accumulates through N 2 ‐fixation, whilst P availability declines, such that N and P co‐limit plant productivity on intermediate‐aged soils (Vitousek and Farrington 1997 ; Laliberté et al. 2012 ). Additionally, while total soil P decreases during pedogenesis, its organic fraction increases and becomes the largest fraction in old soils. In strongly weathered and acidic soils, P can be strongly limiting (Vitousek and Farrington 1997 ; Laliberté et al. 2012 ) and P depletion can be sufficiently severe to cause ecosystem retrogression (Wardle et al. 2004 ; Peltzer et al. 2010 ). Soil chronosequences thus provide a unique opportunity to study changes in plant allocation to different root symbioses with decreasing nutrient availability (Treseder and Vitousek 2001 ). It has been proposed that there is a community‐level shift in the relative importance of different nutrient‐acquisition strategies (specifically, the type of mycorrhizal association) during ecosystem development (Read 1991 ; Lambers et al. 2008 ). In young soils, ruderal nonmycorrhizal strategies and AM associations should be more common (Lambers et al. 2015 ), due to their ability to take up mineral P (Lambers et al. 2012 ; Smith et al. 2015 ). As soils age, a decrease in AM fungi in favor of ECM fungi and ericoid mycorrhizal associations should occur, because the latter can access sorbed and organic forms of P. Finally, in old severely P‐impoverished soils, nonmycorrhizal strategies should become more abundant (Lambers et al. 2008 ; Zemunik et al. 2015 ), given their highly effective strategy to acquire sorbed and organic P (Lambers et al. 2012 ). The validity of this model has been questioned on the basis that vegetation patterns do not follow this model in all chronosequences (Dickie et al. 2013 ). These models have been evaluated by observing changes in plant species composition across soil age (e.g., Zemunik et al. 2015 ), rather than evaluating within‐species shifts in symbiotic associations. The use of plant species capable of forming multiple symbiotic associations allows for a stronger test of these models by controlling for differences in plant host identity. We studied changes in root symbiotic associations (AM, ECM, N 2 ‐fixing nodules) within two plant species that co‐occur across contrasting stages of the Jurien Bay dune chronosequence in southwestern Australia (Laliberté et al. 2012 , 2014 ; Hayes et al. 2014 ). This long‐term dune chronosequence shows a marked decrease in soil P and pH (Laliberté et al. 2012 ; Turner and Laliberté 2015 ), a shift from N to P limitation with increasing soil age (Laliberté et al. 2012 ; Hayes et al. 2014 ), and a high functional diversity in nutrient‐acquisition strategies (Hayes et al. 2014 ; Zemunik et al. 2015 ). We grew seedlings of the two focal species in soils of different ages ( c . 0.1, 1 and 120 ka) in a glasshouse. We hypothesized that within‐species root colonization shifts from AM to ECM with increasing soil age (Lambers et al. 2008 ) and that nodulation in A . rostellifera declines with soil age, reflecting the shift from N to P limitation of plant growth along this chronosequence (Laliberté et al. 2012 ; Hayes et al. 2014 ).",
"discussion": "Discussion Shifts in mycorrhizal colonization Consistent with our hypothesis, root colonization by AM fungi declined with increasing soil age, whereas previous studies have found AM colonization increasing with declining soil P availability (Abbott et al. 1984 ; Bentivenga and Hetrick 1992 ; Treseder and Vitousek 2001 ). However, these studies were conducted at higher soil [P] and across a much smaller soil [P] range (Francis and Read 1994 ) than that along the studied chronosequence (Turner and Laliberté 2015 ). In addition, these studies used species that only form AM, whereas our study focused on species forming multiple associations simultaneously. Furthermore, soil pH decreased and previous studies have shown that AM fungi tend to dominate on young alkaline‐to‐neutral soils (Piotrowski et al. 2008 ; Zangaro et al. 2012 ), and soil pH <5 can decrease AM colonization (Clark 1997 ; Coughlan et al. 2000 ). In our study, pH declined to only 5.8 in the oldest soils, suggesting that pH inhibition likely did not contribute to the effect of soil age on AM colonization. Our results suggest that AM associations are favored in younger soils where most P is in mineral forms (Lambers et al. 2008 ; Turner and Laliberté 2015 ). Root colonization by ECM fungi was about four times greater in the oldest soils than in the youngest soils for both species. Although the oldest soils had a much lower total [P], organic P represented a much larger fraction. Ectomycorrhizal fungi are efficient at accessing organic forms of N and P (Read 1989 ; Antibus et al. 1992 ; Chalot and Brun 1998 ). Consequently, ECM colonization may be related to the organic soil P fraction, consistent with results of Harvey et al. ( 1976 ). Old acidic soils might be better suited for ECM fungi than young alkaline soils (Piotrowski et al. 2008 ; Zangaro et al. 2012 ), as the optimum conditions for ECM fungi are between pH 4 and 5 (Aggangan et al. 1996 ; Yamanaka 2003 ). Young soils in our study exhibited a pH between 5.8 and 8.2, suggesting that the decline in pH contributed to the increase of ECM fungi with increasing soil age. However, we cannot disentangle potential effects of total P from those due to pH, because total P and pH decline simultaneously during pedogenesis. Negative relationships between AM and ECM have been interpreted as competitive exclusion of AM fungi by ECM fungi (Chen et al. 2000 ; Adams et al. 2006 ). Similarly, colonization shifts from AM to ECM with soil depth have been found (Neville et al. 2002 ), with higher ECM colonization in upper soil layers, where organic matter content is greater. In coniferous forest, AM fungi dominate in nutrient‐rich soils with high pH, while ECM fungi dominate in soils with low nutrient availability and lower pH (Nilsson et al. 2005 ). The lack of a relationship between AM and ECM at any soil age in our study suggests that the observed shift from AM to ECM colonization was driven by changes in soil properties, rather than reflecting a direct negative effect of ECM fungi on AM fungi. Shifts in nodule biomass Nodulation in A. rostellifera declined with increasing soil age, likely because plant growth on the oldest soils is limited by the availability of P, rather than N. Nodulation might be constrained in old soils by the relatively high P demand of N 2 fixation (Sprent and Raven 1985 ; Sprent 1999 ; Raven 2012 ). Thus, on old soils, where both N and P availability are extremely low, legumes might acquire N predominantly via ECM, rather than rhizobia. There was no relationship between nodulation and ECM colonization in A. rostellifera once differences in soil age were controlled for. These results differ from those obtained by Diagne et al. ( 2013 ), who found that ECM fungi promote nodulation under P limitation in A. mangium . However, Diagne et al. ( 2013 ) used soils with relatively high P levels (4.8 mg Olsen P kg −1 ), while resin [P] in our study ranged between 0.6 and 3 mg kg −1 (Turner and Laliberté 2015 ). Furthermore, previous studies have shown that a soil pH <4.5 can be detrimental for the two main N 2 ‐fixing rhizobia ( Rhizobium and Bradyrhizobium ; Graham 1992 ; Graham et al. 1994 ). As soil pH in the present study ranged from 8.2 to 5.8, the decrease in nodulation is likely related to nutrient limitation, rather than a low soil pH. Shifts in the type and strength of nutrient limitation Both leaf [N] and leaf [P] reflect the low availability of these nutrients in soils (Laliberté et al. 2012 ; Turner and Laliberté 2015 ). Furthermore, leaf N:P ratio increased more than 10‐fold for A. rostellifera and 20‐fold for M. systena from the youngest to the oldest soils, consistent with shifts from N limitation to strong P limitation of plant productivity along the chronosequence (Laliberté et al. 2012 ; Hayes et al. 2014 ). Leaf N:P increased markedly in A. rostellifera between the youngest and intermediate‐aged soils, while there was no difference between N:P on these two soil ages for M. systena . The change in A. rostellifera was associated with a greater increase in leaf [N], presumably due to its N 2 ‐fixation capacity. Foliar N:P in a N 2 ‐fixing shrub is also low on young soils along a 120 000 year chronosequence in New Zealand (Richardson et al. 2004 ), due to high leaf [N] rather than low leaf [P]. The shifts in mycorrhizal colonization with increasing soil age could be due to changes in inoculum potential, which decreases with increasing soil age for AM fungi, but increases with soil age for ECM fungi (Piotrowski et al. 2008 ; Zangaro et al. 2012 ). However, such changes in inoculum potential might be related to longer‐term feedback between plants and soil biota that ultimately depend on soil nutrient availability. Additionally, soils in this study were sieved and dried at nondetrimental temperatures (Lucas et al. 1992 ), yet this could have potentially removed fungal species that colonize through hyphae. Future experiments should aim to disentangle the role of such biotic and abiotic effects on the balance of multiple symbioses, to assess the effects of soil abiotic properties and inoculum potential independently. In conclusion, our results show within‐species shifts between different root symbiotic associations during long‐term soil and ecosystem development, consistent with those predicted by Read ( 1991 ) and Lambers et al. ( 2008 ). This might be associated with a shift from N to P limitation of primary plant productivity, soil pH or inoculum potential (Nilsson et al. 2005 ; Zangaro et al. 2012 ). Our study supports the hypothesis that the importance of different mycorrhizal types changes with soil age (Lambers et al. 2008 ). Our results on intraspecific shifts in nutrient‐acquisition strategies complement those of a recent study along the same chronosequence showing that, at the community level, ECM plants become more abundant as soils age (Zemunik et al. 2015 ). Further work on within‐species shifts in symbiotic associations and their functional significance is needed to better understand the role of mycorrhizal fungi during long‐term ecosystem development (Dickie et al. 2013 )."
} | 4,188 |
37514432 | PMC10383247 | pmc | 6,169 | {
"abstract": "Robust membrane materials with high efficiency have attracted extensive attention in oil/water separation. In this work, carbon particles via candle combustion were firstly adsorbed on the surface of stainless steel meshes (SSMs), which formed a thin hydrophobic coating, and a rough structure was then constructed through chemical vapor deposition and high temperature calcination, with the resultant SSM surface wrapped with uniform silica coating possessing the characteristic of superoleophobicity underwater. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and X-ray powder diffraction (XRD) were used to characterize the modified SSMs. The prepared SSMs were superhydrophilic in air, and they had superoleophobicity underwater (157.4°). The separation efficiency of five oil/water mixtures was above 98.8%, and the separation flux was 46,300 L·m −2 ·h −1 . After it was immersed in 1 mol/L NaOH, 1 mol/L HCl and 3.5 wt% NaCl for 24 h, respectively, the efficiency was still above 97.3%. Further immersion in the solution of dopamine and octadecylamine resulted in the transformation of superhydrophililc/superoleophobicity-underwater SSMs to superhydrophobic SSMs, and the resultant SSMs with reverse surface wettability was also used for the oil/water separation with good separation efficiency and separation flux.",
"conclusion": "4. Conclusions In this study, superoleophobic SiO 2 /SSM was prepared by sequentially depositing candle ash and silica nanoparticles on the surface of the SSM, followed by calcination at 600° for about 2 h. In addition, the SSM still maintained good superhydrophilic-underwater superoleophobic properties in harsh environments. Further chemical modification for the underwater superoleophobic SSMs created a superhydrophobic material. Both modified SSMs exhibited high-efficiency of oil/water separation, with a remarkable separation efficiency of about 98% or so, and a water permeate flux of more than 2.6 × 10 4 L m −2 h −1 . This work has developed a facile and versatile process for the fabrication of oil/water separation membranes without the use of environmentally harmful fluorinated substances.",
"introduction": "1. Introduction The leakage of petrochemical products and the discharge of oily wastewater can cause serious ecological and environmental problems, so it is a serious challenge to select suitable methods and materials to treat oily wastewater quickly and efficiently [ 1 , 2 , 3 ]. At present, some traditional methods, such as adsorption [ 4 ], air flotation [ 5 ], flocculation [ 6 ], biological treatment [ 7 ], electrochemical treatment [ 8 , 9 ], and membrane filtration [ 10 , 11 , 12 ], are often applied for oil/water separation, but most of these separation methods have shortcomings, such as excessive energy consumption, low separation efficiency, long separation cycles, poor chemical and mechanical stability, and, sometimes, even secondary contamination [ 13 , 14 ]. With the rapid development of surface science and bio-nanotechnology, bioinspired materials with superwettability have been increasingly used in oil/water separation. The special properties of surface wettability mainly contain superoleophobicity, superhydrophilicity, superlipophilicity, and superhydrophobicity [ 15 ]. To achieve oil/water separation, it is necessary to have different wettability for water and oil, i.e., superhydrophobic/superoleophilic or super-hydrophilic/underwater superoleophobic. From the energy point of view, the surface tension of water is around 72.8 mN/m, while the surface tension of common organic phases is generally in the range of 20~40 mN/m. Therefore, materials that are hydrophilic in air must have a high surface energy, which determines that they are also necessarily lipophilic in air. However, if we want to use the hydrophilic material for oil/water separation, the special infiltration properties with superhydrophilicity in air and superoleophobicity underwater should be endowed [ 16 ]. Currently, oil/water separation is mainly achieved by super-hydrophobic/super-oleophilic materials, by which water is blocked but oil penetrates. In contrast, oil is blocked but water penetrates for superhydrophilic/underwater superoleophobic materials. Their separation properties are determined by the chemical composition of the surface and its microscopic geometry, and, often, the superwettability of the membrane material has to be modulated by constructing rough surfaces [ 17 ]. Superhydrophobic/superoleophilic materials have to treat the micro/nano-structures with low surface energy substances in order to repel water highly, and the hydrophilic chemical composition of the micro/nano-structures should be tailored in order to construct the superhydrophilic surfaces for the preparation of superhydrophilic/underwater superhydrophobic materials. Chen et al. prepared superhydrophobic SSM films by chemical etching and solution immersion. The separation efficiency of the mesh membrane modified with low surface energy substance of 1,2-hydroxystearic acid can be as high as 95.65% for petroleum ether and water, and the separation efficiency of the mesh membrane can still be maintained above 90% in weak acidic and alkaline as well as saline environments [ 18 ]. Ding et al. used the prepared hydrophobic and lipophilic SiO 2 gels deposited on the surface of the SSM for the separation of kerosene and water, and the separation efficiency could still reach 97% after 40 cycles of oil/water separation [ 19 ]. Li et al. prepared a superhydrophobic SSM by spraying hydrophobic nano SiO 2 on an SSM with carbon particles, which was adsorbed due to candle combustion. During the oil/water separation process, the surface nanoparticles were easily dislodged, and they easily lost their hydrophobic and lipophilic properties due to the poor attachment of the deposited carbon particles and hydrophobic nano-SiO 2 to the smooth SSM [ 20 ]. Lei et al. used epoxy resin and fluorinated nano-graphite flakes to modify the surface of the SSM, and they prepared a superhydrophobic steel mesh with excellent separation of oil/water mixtures of n -hexane, n -decane, m-xylene, diesel, and methylene chloride, all with permeate fluxes above 41,000 L/(m 2 ·h) and separation efficiencies up to 99.9% [ 21 ]. Inspired by the natural phenomenon that fish scales are not adhered by oil in water, Jiang et al. [ 22 ] coated a layer of hydrophilic polyacrylamide hydrogel (PAM) on SSM and obtained a surface with superhydrophilic-underwater superoleophobic properties, which successfully carried out the separation of oil/water mixtures. Cao et al. prepared superhydrophilic/underwater superhydrophobic membranes by in situ curing TiO 2 nanoparticles and polyvinylpyrrolidone (PVP) onto SSM, and the contact angle of oil in water reached 160° and the oil/water separation efficiency was greater than 99.5%. The maximum membrane flux was 8422.5 L/(m 2 ·h) [ 23 ]. Vollmer’s group used soot as a template to prepare robust transparent superamphiphobic glasses, and their work was published in the main issue of Science [ 24 ]. The authors first constructed a porous coating from candle ash on the glass slide and then modified the surface by chemical vapor deposition (CVD) [ 25 , 26 ], followed by calcination at 600 °C for about 2 h. After the entrapped carbon nanoparticles were removed, a robust superamphiphobic coating was obtained after further CVD with a low-surface energy substance. Referring to this method, Chao et al. prepared an identical glass flake and focused on investigating the anti-bacterial properties of the coating [ 27 ]. In this paper, a high strength, economical, and easily available SSM (SSM) is used as the substrate and a certain thickness of soot is adsorbed on the surface of the SSM through the candle combustion process, and silica is then uniformly deposited on the SSM surface by vacuum-assisted CVD of tetraethoxysilane (TEOS). The sol-gel method was catalyzed by ammonia solution [ 28 , 29 ], and the formed hybrid carbon/silica network was further calcinated, which led to the removal of carbon and intimate attachment of the silica network on the surface of SSMs. The resultant superhydrophilic/underwater superoleophobic SSMs were further modified to the superhydrophobic SSMs by the solution immersion of octadecylamine. Both SSWs with different surface wettability were successfully used for oil/water separation, and their separation performance and efficiency were investigated in detail.",
"discussion": "3. Results and Discussion 3.1. Microscopic Morphology and Composition Analysis SEM was used to characterize the surface morphology of pristine SSM, SiO 2 /SSM-1. As shown in Figure 2 a, the pristine SSM surface was smooth without obvious attached substances. Figure 2 b showed the soot layer, and the metal mesh induced the incomplete combustion of the candle and produced a large number of soot particles, which uniformly covered the surface of the SSM. The SSM surfaces after CVD for 16 h, 24 h, and 36 h and after calcination are illustrated in Figure 2 c,d,f, respectively. A small amount of nanoparticles were deposited on the SSM surface with a deposition time of 16 h, and the mesh aperture was still relatively obvious at this time. After 24 h deposition, the mesh size was obviously reduced, and the thickness of the silica nanoparticles covered on the surface was increasing. As the deposition time increased to 36 h, the presence of the mesh pores was almost invisible, and the thick deposition of SiO 2 particles may have led to looser attachment on the SSM surface. Therefore, a deposition time of 24 h was chosen for the next application. EDS results showed the elemental distribution of SSM before and after modification (see Figure 3 a–c). Compared with the pristine SSM that O, Cr, Fe, and Ni were distributed uniformly on the SSM skeleton, with atomic ratios of 5.09%, 18.87%, 69.02%, and 7.73%, respectively, the comparative content of the four elements significantly changed, with atomic ratios of 64.55%, 2.17%, 6.57%, and 0.56%. The content of O was significantly increased, and, at the same time, the element Si appeared, which indicated that SiO 2 is generated on the surface of the SSM. The surface chemical bonds of the pristine SSM and SiO 2 /SSM-1 were further analyzed using XPS ( Figure 3 (d1–d3)). The positions of the characteristic peaks of the elements revealed the characteristic peak of O 1s at the binding energy of 532.65 eV, the characteristic peak of C 1s at the binding energy of 284.81 eV, and the characteristic peak of Si 2P at the binding energy of 103.51 eV. Compared with the pristine SSM, the SiO 2 /SSM has more intensive peaks of both O and Si elements, which proves the presence of SiO 2 . In the high-resolution XPS spectrum of O 1s ( Figure 3 (d2)), the peak fitted at 533.7 eV corresponded to the Si-O bond, and in the high-resolution XPS spectrum ( Figure 3 (d3)), the fitted peak corresponded to the Si 4+ state, thereby confirming the generation of SiO 2 . It is also difficult to observe the C peaks on the prepared SiO 2 /SSM-1, which indicates that the high temperature calcination removed the carbon particles thoroughly [ 23 ]. Herein, CVD led to the reaction of hydrolysis and condensation of TEOS, and the form of silica was similar to a Stöber reaction. Generally, the mass of silica layers deposited on the mesh in the absence of candle ash for about 24 h was calculated to be about 0.18 mg/cm 2 . 3.2. Wettability of the SSM Surface before and after Modification The wettability of the SSM before and after modification was analyzed using a contact angle tester. The water contact angle (WCA) and underwater oil contact angle (UWOCA) were both measured. As illustrated in Figure 4 , the WCA in air on the pristine SSW was 103° ± 2.5° ( Figure 4 a) and the UWOCA of n -octane was close to 0° ( Figure 4 b), which indicated its typical superhydrophilic/underwater superoleophobic property. In air, the modified SSM was superhydrophilic, and when water droplets contacted the SiO 2 /SSM surface, they quickly spread on the mesh, reaching a CA of 0° in 112 ms ( Figure 4 d), whilst the average CA of n -octane underwater for three measurements was 157.4° ± 1.2° ( Figure 4 c). This underwater superoleophobic structure is beneficial for oil/water separation [ 31 , 32 ]. In order to test the underwater superoleophobicity of different oils on the modified SSM, five organic solvents, namely, hexane, n -octane, isooctane, petroleum ether, and carbon tetrachloride, were selected for the test. As listed in Table 1 , the density of carbon tetrachloride is 1.594 g/mL and can represent heavy oil and the other four organic solvents in the density between 0.65–0.703 g/mL can stand for light oil, and the surface tension of all five solvents is less than 30 mN/m. As illustrated in Table 1 , the average values of UWOCA ( n = 3) were 157.3°, 154.2°, 157°, 152.6°, and 148.5° for n -hexane, n -octane, iso -octane, petroleum ether, and carbon tetrachloride, respectively. This indicated that the modified SSM behaved obviously underwater in terms of superolephobicity and that it has potentially universal applicability for oil/water separation. Herein, it should be noted that the SSM without the soot layer did not exhibit underwater superoleophobicity in the presence of CVD for about 24 h. 3.3. Oil/Water Separation Performance for SiO 2 /SSM-1 An immiscible solution of oil and water phases was poured onto the pre-wetted SiO 2 /SSM-1, which is fixed and sandwiched between two glass tubes. The oil/water separation process is shown in Figure 5 . For the oil phase, n -hexane, n -octane, iso -octane, petroleum ether, and carbon tetrachloride was dyed red with Sudan III, respectively, and it was dyed blue with methylene blue for the water phase. The two phases were mixed according to V oil /V water = 1/1, and were then poured into the glass tube from the top. When all of the deionized water entered the collection bottle below, timing was stopped and the spent time started being recorded. The whole separation process was carried out only under the action of gravity. Water penetrated quickly, and the oil was blocked above the SiO 2 /SSM-1. The separation mechanism can probably be attributed to the fact that the superhydrophilic surface of SiO 2 /SSM preferentially attracted the water phase in the three-phase system of oil/water/solid, and that the highly rough surface resulted in a quite small area fraction of the solid. Water molecules were tightly trapped in the rough SiO 2 /SSM-1 micro/nanostructures, which allowed the water phase to pass through the mesh by its gravity quickly, and the formed water barrier layer repelled the oil penetration. The separation efficiency was calculated by the following formula: η = m 1 m 0 × 100 % Here, m 0 and m 1 are the masses of water (g) before and after separation, respectively. It can be seen that different mixtures of oil and water are separated efficiently with high separation fluxes using the SiO 2 /SSM [ 33 ]. Figure 6 shows the separation efficiencies and permeate fluxes of the different light oil and water mixtures. The separation flux was calculated by the following equation: J = V/(S∙∆t) \nwhere J(L·m −2 ·h −1 ) is the flux, V(L) is the filtrate volume, S(m 2 ) is the effective area, and ∆t(h) is the permeation time. The performance of oil/water separation was investigated with a separation efficiency higher than 98.1%, and permeation fluxes of water were in the range of 38,000 L·m −2 ·h −1 ~46,300 L·m −2 ·h −1 . Moreover, after 20 cycles of separation, the separation efficiency was still greater than 97.3%, thus demonstrating its excellent stability and durability properties. In the practical application of oil/water separation, the separation material usually needs to be used in harsh environments, so the chemical durability of the material is very important [ 34 , 35 , 36 , 37 , 38 , 39 ]. The fabricated SiO 2 /SSM-1 was thus immersed in 1 mol/L NaOH, 1 mol/L HCl, or 3.5 wt% NaCl (simulated seawater) solution, respectively. The UWOCA values of n -octane on the SiO 2 /SSM-1 were still 152°, 156°, and 154° with an oil/water separation efficiency greater than 95% after solution immersion for about 24 h. This demonstrates that the superhydrophilic underwater superoleophobic properties were not destroyed for our prepared robust SSMs. 3.4. Oil/Water Separation Performance for SiO 2 /SSM-2 After hydrophobic modification with a low-surface energy material of octadecylamine, the prepared SSMs with hierarchical structures possessed superhydrophobicity, which benefited the oil/water separation. As shown in the Figure 7 , the water droplet (blue-dyed by methylthionine chloride) was seated spherically on the superhydrophobic surface of SSM-2, with a WCA of 153.2°. Additionally, the performance of oil/water separation for SiO 2 /SSM-2 was investigated in Figure 8 , with a separation efficiency higher than 97.9 ± 2.1% and permeation fluxes of water in the range of 26,280 ± 117 L·m −2 ·h −1 ~55,800 ± 146 L·m −2 ·h −1 ."
} | 4,276 |
32039180 | PMC6985040 | pmc | 6,171 | {
"abstract": "The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D - and L -glutamic acid monomers, currently relies on L -glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus , offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h −1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D -xylose/ D -glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D -xylose/ D -glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.",
"introduction": "Introduction Poly-γ-glutamic acid (γ-PGA) is a biopolymer consisting of D - and L -glutamic acid monomers. The monomers are linked via amide linkages between the γ-carboxyl and the amino group of monomers γ-PGA is a non-toxic, biodegradable polymer. Due to these properties, γ-PGA is suitable for various applications in industrial fields including bioremediation, food sector, and medical use. The chemical synthesis of γ-PGA is complex since glutamate has two carboxyl groups. Thus, industrial production of this polymer is solely based on bacterial fermentation. The cost of production is hindering many applications of biopolymers (Kreyenschulte et al., 2014 ). Therefore, lowering the production cost by utilization of cheap substrates derived from plant biomass becomes an important task for a more efficient bioprocess of γ-PGA. One of the suitable substrates is xylose, the second most abundant carbohydrate in nature and the main pentose of hemicellulose in plant biomass. Depending on the origin of the hemicellulose, xylose makes up 90% of the hemicellulose (Saha, 2003 ). The production of γ-PGA depends on the PGA synthetase catalyzing the polymerization reaction of L - and D -glutamic acid monomers and exporting the γ-PGA to the extracellular space. The glutamic acid precursors can be either de novo synthesized or imported from the medium. For de novo synthesis of glutamate from glucose, glucose is converted to acetyl-CoA by glycolysis, which then enters the TCA cycle to form 2-oxoglutarate. The glutamate synthase encoded by gltAB catalyzes the conversion of 2-oxoglutarate and glutamine to L -glutamate (Bohannon et al., 1985 ; Belitsky et al., 2000 ). This reaction is NADPH dependent. In absence of glutamine, L -glutamate can be synthesized from 2-oxoglutarate and ammonia by glutamate dehydrogenase. L -glutamate can be converted to D -glutamate directly by glutamic acid racemase or indirectly via D -amino acid aminotransferase (Ashiuchi, 2010 ). Besides glucose, B. subtilis 168 can also grow on xylose as sole carbon source. In B. subtilis 168, xylose is taken up via the arabinose transporter. Xylose is metabolized by a combination of the xylose isomerase pathway and the pentose phosphate pathway (PPP). The genes encoding the xylose isomerase and xylulokinase are xylA and xylB , respectively. These enzymes convert xylose to xylulose-5-phosphate, which is an intermediate of the PPP. In other bacteria as Pseudomonas taiwanensis VLB120 and Caulobacter crescentus the alternative Weimberg pathway for the conversion of xylose to 2-oxoglutarate has been described (Weimberg, 1961 ; Stephens et al., 2007 ; Köhler et al., 2015 ). The C. crescentus xyl operon including the five genes xylXABCD was shown to encode the enzymes for this linear D -xylose oxidative pathway. D -xylose is converted via D -xylono-1,4-lactone, xylonate, 2-keto-3-deoxy- D -xylonate, and 2-oxoglutarate-semialdehyde to 2-oxoglutarate (Stephens et al., 2007 ). The Weimberg pathway was successfully integrated into Pseudomonas putida S12 to enable the utilization of xylose as carbon source (Meijnen et al., 2009 ). Whereas, for the wild-type strain xylose is catabolized via the PPP, the integration of the Weimberg pathway to B. subtilis and simultaneous deletion of the native xylose degradation pathway likely directs the carbon flux to 2-oxoglutarate with a theoretical carbon yield of 100% (see Figure 1 ). Figure 1 Metabolic pathways for γ-PGA production with xylose utilization. The isomerase pathway (blue) is natively present in Bacillus subtilis . The genes xylA and xylB encode the xylose isomerase and xylulokinase, respectively. These enzymes convert xylose to xylulose-5-phosphate, a pentose phosphate pathway (PPP) intermediate. The Weimberg pathway as present in Caulobacter crescentus (green) consists of five genes, xylA, xylB, xylC, xylD , and xylX . It is a linear pathway converting xylose to 2-oxoglutarate. In this study, γ-PGA production with xylose as substrate is demonstrated. The core stoichiometric model for growth and γ-PGA formation (Zhu et al., 2013 ) was extended with metabolic pathways for xylose utilization. The model was used to calculate the γ-PGA production rate and yield for different substrate compositions. Metabolite measurements were carried out to validate the hypothesized higher precursor supply for xylose metabolism via the Weimberg pathway. Using the engineered strains, γ-PGA was produced with xylose as sole carbon source as well as with glucose/xylose mixtures. In summary, we present an alternative γ-PGA production process that can be the basis for future cost efficient and eco-friendly γ-PGA production.",
"discussion": "Discussion Integration of the C. crescentus xylXABCD genes was demonstrated to enable bacteria such as Pseudomonas (Meijnen et al., 2009 ) and Corynebacterium glutamicum (Radek et al., 2014 ) to grow on xylose as sole carbon and energy source. The direct conversion of xylose to the C5-compound 2-oxoglutarate without carbon loss is beneficial for γ-PGA production as well as for amino acids derived from 2-oxoglutarate. However, the reported maximal growth rate with μ = 0.21 h −1 for P. putida S12 (Meijnen et al., 2009 ) and μ = 0.07 h −1 for C. glutamicum (Radek et al., 2014 ) were low. In both cases, plasmid-based expression of the enzymes of the Weimberg pathway was used. The genomic integration of xylXABCD in B. subtilis WB led to a growth rate of 0.43 h −1 ( Table 2 ). The genomic integration circumvents the metabolic burden of plasmid replication. The replacement of the native B. subtilis xylose operon allowed for the regulation of expression by the native B. subtilis xylose promoter. The resulting growth rate was even higher than for the wild-type B. subtilis using the isomerase pathway. In P. putida and C. glutamicum , the accumulation of xylonate was observed in the supernatant. Xylonate accumulation strongly indicates that the xylonate dehydratase reaction converting xylonate to 2-keto-3-desoxy-xylonate is the rate-limiting step of the pathway. The inefficient conversion of xylonate is likely one factor for the reduced growth rates in these bacteria. For B. subtilis WB, the higher growth rate indicates a sufficient activity of all enzymes. Nevertheless, a bi-phasic (diauxic) growth with a lag phase between the two growth phases was observed. Here, intracellular metabolite measurements revealed the accumulation of TCA intermediates such as malate and fumarate. This indicates that the conversion of malate and oxaloacetate to pyruvate and phosphoenolpyruvate are rate-limiting steps in B. subtilis WB. The two xylose utilization pathways, xylose isomerase and Weimberg pathway were compared with regard to the precursor supply for γ-PGA production. In contrast to the hypothesized higher 2-oxoglutarate supply for the Weimberg mutant, the concentration of most TCA cycle intermediates was higher for the xylose isomerase pathway. The metabolite measurements indicate a higher activity TCA cycle when xylose is converted to 2-oxoglutarate. This results in lower metabolite concentrations for TCA cycle intermediates. These results are consistent with the metabolome analysis for γ-PGA-producing B. licheniformis (Mitsunaga et al., 2016 ). For higher fluxes toward γ-PGA when glycerol is used as carbon source, the precursor concentrations of citrate, isocitrate, and 2-oxoglutarate are significantly lower than for glucose with a lower γ-PGA production rate. The higher TCA cycle activity was demonstrated to result in higher γ-PGA synthesis for the Weimberg mutant. Several studies demonstrated an efficient γ-PGA synthesis with glutamic acid as additional carbon source (Cromwick et al., 1996 ; Richard and Margaritis, 2003 ). The majority of these studies involve wild-type γ-PGA producers that require glutamate for γ-PGA synthesis. For glutamate-independent production of γ-PGA the media commonly contain citric acid to enable high γ-PGA titers (Kongklom et al., 2017 ). In all cases, the γ-PGA production is greatly increased when direct glutamate precursors are used as substrates. The direct conversion of xylose to 2-oxoglutarate implemented in this study presents an alternative solution for higher precursor supply from cheaper substrates. Since up to 25% of lignocellulosic biomass are made up from pentoses (Lee, 1997 ), their use is essential to efficiently produces bio-based products. When glucose and L -glutamate were used as carbon sources, only 6–9% of the glutamate that was incorporated into γ-PGA was de novo synthesized from glucose (Yao et al., 2010 ). Therefore, an increase in the initial glucose concentration mainly resulted in higher biomass formation instead of higher γ-PGA synthesis. Hence, the utilization of two carbon sources may be beneficial if one carbon source is used for growth and energy production and another one is used for γ-PGA precursor supply. The investigation of the theoretical flux distributions emphasized this hypothesis. A higher theoretical γ-PGA production rate was observed for glucose and xylose mixtures compared to xylose alone. For substrate mixtures, glucose was converted to biomass precursors and used as energy source. Xylose was converted to glutamate without carbon loss. However, the consecutive uptake of the two substrates is not considered in the theoretical flux distribution. Further engineering may focus on the utilization of both carbon sources (Wu et al., 2016 ). In this study, the integration of promoter PX43 for PGA synthetase aimed at the discrimination of growth phase on glucose and production phase on xylose. Since the viscosity started increasing before glucose depletion, the PGA synthetase was expressed despite of the cre site for glucose repression. Furthermore, the theoretical flux distributions strongly depend on the growth rate and substrate uptake rate. A growth rate of 0.6 h −1 as used for the FBA was only proven to be true for growth on glucose. The changing growth rate throughout the cultivation was not considered. In this study, the Weimberg pathway was successfully implemented into B. subtilis to achieve high γ-PGA production from biomass-derived substrates. Thereby, the carbon loss for utilization of xylose as substrate was minimized. The use of a mixture of glucose and xylose enabled the γ-PGA synthesis with a yield of 0.26 C-mol/C-mol. The yield may further be improved by strain and process development to achieve resource efficient γ-PGA production from biomass-derived substrates."
} | 3,026 |
35254257 | PMC8963879 | pmc | 6,172 | {
"abstract": "During colony growth, complex interactions regulate the bacterial orientation, leading to the formation of large-scale ordered structures, including topological defects, microdomains, and branches. These structures may benefit bacterial strains, providing invasive advantages during colonization. Active matter dynamics of growing colonies drives the emergence of these ordered structures. However, additional biomechanical factors also play a significant role during this process. Here, we show that the velocity profile of growing colonies creates strong radial orientation during inward growth when crowded populations invade a closed area. During this process, growth geometry sets virtual confinement and dictates the velocity profile. Herein, flow-induced alignment and torque balance on the rod-shaped bacteria result in a new stable orientational equilibrium in the radial direction. Our analysis revealed that the dynamics of these radially oriented structures, also known as aster defects, depend on bacterial length and can promote the survival of the longest bacteria around localized nutritional hotspots. The present results indicate a new mechanism underlying structural order and provide mechanistic insights into the dynamics of bacterial growth on complex surfaces.",
"introduction": "Introduction Bacterial colonization and invasion are collective phenomena. These processes are regulated through a complex interplay of physical and biological interactions in a crowded population. Bacterial morphology, hydrodynamics, surface topology, and topography markedly alter growth mechanisms, morphology, and overall competition among bacteria ( Grant et al., 2014 ; Su et al., 2012 ; Volfson et al., 2008 ; Warren et al., 2019 ; Cho et al., 2007 ; Smith et al., 2017 ). Elucidation of the factors regulating collective bacterial growth and their competition is essential to enhance our understanding of evolutionary dynamics, bacterial infection, and the progression of inflammatory diseases. A characteristic feature of bacterial colonization is the formation of large-scale order. Rod-shaped bacteria display nematic alignment on surfaces, wherein localized stress, surface friction, and elasticity trigger the formation of ordered domains and lead to the emergence of topological defects ( Doostmohammadi et al., 2016 ; Dell’Arciprete et al., 2018 ; Doostmohammadi et al., 2018 ; You et al., 2018 ; You et al., 2021 ) and various types of self-assembled structures, including edge fingerings ( Farrell et al., 2013 ) and vertical structures ( Beroz et al., 2018 ; Hartmann et al., 2019 ). In particular, ±½ topological defects are the typical orientational singularities observed among growing bacterial colonies and biofilms ( Doostmohammadi et al., 2016 ; Doostmohammadi et al., 2018 ; You et al., 2018 ; Yaman et al., 2019 ). These topological defects have biological significance and regulate stress distribution across the structure, alter the physiology of the cells ( Saw et al., 2017 ), and could control entire morphology; eventually, these effects trigger the formation of fruiting bodies ( Copenhagen et al., 2020 ) and bacterial spores in biofilms ( Yaman et al., 2019 ). Liquid crystal theory has successfully predicted the dynamics of these defects; - 1 2 defects are stationary whereas + 1 2 defects are generally motile ( Shankar and Marchetti, 2019 ; DeCamp et al., 2015 ; Giomi et al., 2013 ). Another interesting structural order in bacterial colonies is anchoring, where the bacteria are tangentially oriented along the edge of the colony ( Su et al., 2012 ; Doostmohammadi et al., 2016 ; Dell’Arciprete et al., 2018 ). In this study, we assess the orientational dynamics of a crowded bacterial population competing for limited space. Unlike regular expanding colonies, if growing bacteria surround a closed area, domains of inward growth are formed. Under these conditions, entire mechanical interactions differ and lead to the formation of asters, formed as radially aligned +1 topological defects. With only a few exemptions ( Maroudas-Sacks et al., 2020 ; Meacock et al., 2021 ), higher-order topological defects ( Thijssen and Doostmohammadi, 2020 ; Shankar et al., 2018 ) are not commonly observed in extensile active matter systems, including growing bacterial colonies. These defects only appear under external modifications such as stress ( Rivas et al., 2020 ), confinement ( Duclos et al., 2016 ; Opathalage et al., 2019 ), and flow ( Martínez-Prat et al., 2019 ). Our results also reveal that velocity profile is an important factor controlling the emergence of these radially aligned structures. Furthermore, we investigate the invasive advantages of this orientation for competing bacterial strains of different lengths. Inward growth is commonly observed in various biological systems. During wound healing ( Basan et al., 2013 ), cancer cell growth ( Lee et al., 2017 ; Vader et al., 2009 ), and retina development ( Than-Trong and Bally-Cuif, 2015 ; Azizi et al., 2020 ), similar dynamic mechanisms are underway. Our results may provide novel mechanistic insights into these dynamics, particularly on the physical conditions for radial structural alignments during these complex growth processes.",
"discussion": "Discussion Radially aligned structures can be considered as a +1 aster defect. These are ubiquitous topological structures observed in biological ( Roostalu et al., 2018 ; Ross et al., 2019 ; Kruse et al., 2004 ; Julicher et al., 2007 ) or synthetic ( Sokolov et al., 2019 ; Snezhko and Aranson, 2011 ) active matter systems. For instance, microtubules can form nematic alignment or asters during mitosis, depending on the extensile or contractile activity. Bacterial colonies can be considered an extensile active material platform, generally supporting the formation of only ±½ topological defects. This study shows that stable radially aligned, aster structures can also emerge during inward growth. In particular, we report the critical role of the colony velocity profile during this process, which depends on numerous factors. Although the bacterial growth rate is constant throughout the colony, growth geometry, confinement, or boundary conditions can alter the velocity profile. Together, these biomechanical interactions change the bacterial orientation and stability, thus generating ordered structures. Different types of ordered structures have been observed in bacterial biofilms ( Yan et al., 2016 ) and 3D colonies ( Warren et al., 2019 ). Furthermore, we believe that the velocity profile of growing structures on flat surfaces plays a significant role in bacterial alignment. Future studies are required to investigate the contribution of these effects. We should emphasize that inward-growing bacterial colonies and wrinkling thin circular sheets have geometric similarities ( Davidovitch et al., 2011 ). In these elastic circular objects, under axisymmetric tensile load, azimuthal stress (hoop stress, σ θ θ ) show transition from tensile to compressive profile which eventually creates radial wrinkling pattern below critical radius. However, unlike elastic objects, growing bacterial colonies can only develop compressive stress due to negligible attractive force between bacteria. Experimental measurement of internal stress could provide more details, but it remains challenging. We noticed that the packing fraction of the bacteria shows a correlated profile ( Figure 2—figure supplement 2 ). However, particularly for aligned bacteria, it is still very difficult to extract this information. In the future, new molecular probes could be useful for the experimental measurement of accumulated stress in the bacterial colonies ( Chowdhury et al., 2016 ; Prabhune et al., 2017 ). Finally, this study reveals the potential biological significance of radial alignment during the invasion. These ordered structures provide additional advantages and promote the survival of the longest bacteria. These results link the orientational properties and competition dynamics of bacterial colonies. Our findings are of potential relevance for the understanding of complex dynamics of bacterial infections and the progression of inflammatory diseases."
} | 2,055 |
27744069 | null | s2 | 6,174 | {
"abstract": "Mussel adhesive moiety, catechol, has been utilized to design a wide variety of biomaterials. However, the biocompatibility and biological responses associated with the byproducts generated during the curing process of catechol has never been characterized. An in situ curable polymer model system, 4-armed polyethylene glycol polymer end-capped with dopamine (PEG-D4), was used to characterize the production of hydrogen peroxide (H Remarkable underwater adhesion strategy employed by mussels has been utilized to design a wide variety of biomaterials ranging from tissue adhesives to drug carrier and tissue engineering scaffolds. Catechol is the main adhesive moiety that is widely incorporated to create an injectable biomaterials and bioadhesives. However, the biocompatibility and biological responses associated with the byproducts generated during the curing process of catechol has never been characterized. In this manuscript, we design a model system to systemically characterize the release of hydrogen peroxide (H"
} | 256 |
35497842 | PMC9049918 | pmc | 6,176 | {
"abstract": "Although various filtration materials with (super)wetting properties have been fabricated for effective oil/water separation, eco-friendly and low-cost materials are still highly desired. This work details the facile preparation of efficient oil–water separation papers with superhydrophobic properties that successfully combine micro/nanoscale hierarchical particles and low surface energy components with porous substrates. The superhydrophilic papers were coated with a polydopamine layer and then immersed in the mixture of polydimethylsiloxane (PDMS) and hydrophobic-silica nanoparticles. The resultant paper can separate oil–water mixtures under gravity driving conditions, where heavy oil penetrates through the sample and water is collected on the surface. And the as-prepared sample had favorable separation efficiency (>99%). More importantly, the oil flux almost remained at the original value after 10 cycles, indicating excellent recyclability. In addition, the as-prepared paper exhibits good stability in acidic, alkaline and salty media.",
"conclusion": "Conclusions In summary, we have developed a simple strategy for fabricating superhydrophobic paper having the capacity to separate oil–water mixtures under gravity driving conditions. The nascent papers were coated with a PDA layer and then immersed in the mixtures of PDMS and hydrophobic-SiO 2 nanoparticles. It is shown that the micro/nanoscale hierarchical particles of PDMS–SiO 2 /PDA covered on the porous substrate. The water droplets can rebound on the modified low energy surface without wetting or even residual. Upon dumping oil–water mixture into the surface, heavy oil quickly penetrates through the sample, while water is retained on the surface. The separation efficiency is larger than 99%. Furthermore, the modified paper shows excellent recyclability and stability in acidic, alkaline and salty. We believe that such a high-efficiency, low-cost and eco-friendly paper has great potential for the practical application in solve the oily wastewater.",
"introduction": "Introduction Effective separation for oil–water has drawn significant worldwide attention because of the weaknesses in freshwater supply for the rapidly growing global human population. This kind of wastewater comes from a variety of sources such as crude oil production and refineries, petrochemical, pharmaceutical, metal processing, and textile production industries, and oil spill accidents from storage tanks or transport facilities. They often contain various toxic substances such as phenols, petroleum hydrocarbons, polyaromatic hydrocarbons, metal ions, and radioactive elements that possess mutagenic and carcinogenic risks to plants, animals and human beings. 1–3 A variety of conventional methods such as gravity or density separation, burning, skimming, centrifugation, coagulation, adsorption, etc. , have been invented to separate oil–water. 4–11 However, these methods are not completely satisfactory. For instance, the coagulation and gravity separation techniques are low efficiency and multistage ( Fig. 1A ). Fig. 1 Schematic illustration of oil/water separation processes by the coagulation and gravity separation techniques (A) and the fabrication for the film of PDMS–SiO 2 /PDA/paper and its application for oil/water separation (B). Recently, filtration techniques have been proved to be one of the best methods for effective separation of oil–water, which only allows a selected phase (either oil or water) to penetrate, while preventing the other phase from passing through. Various polymer membranes, 12–16 metal meshes, 17–20 nanofibers, 21,22 graphene oxide/carbon nanotube filters 23–26 with selective wettability have been fabricated via phase separation, electrochemical deposition, chemical vapor deposition, dip coating, etc. Now, superhydrophobic/superoleophilic “oil-removing” materials and underwater superoleophobic “water removing” materials have received a great deal of attention. Hydrophobic polydimethylsiloxane (PDMS) is one of the most important materials in the preparation of superhydrophobic and superoleophobic surfaces based on its very low surface tension and self-roughening. 27–32 Dopamine, the most renowned catecholamine, is able to undergo self-polymerization under oxidizing conditions, creating a bioinspired surface-adherent polydopamine (PDA) coating on almost all kinds of organic and inorganic surfaces. 33–36 Moreover, PDA chains incorporate many functional groups such as catechol, amine, and imine, that can serve as the reactive sites with desired molecules and the anchors for the loading of transition metal ions via bidentate coordination, hydrogen bonding, Michael addition or Schiff base reactions for efficient oil/water separation. 34,37–42 Lee group modified anodic aluminum oxide (AAO) membranes by adhesion mechanisms of PDA and soft-lithographic technique. The modified superhydrophobic surface showed high-water adhesion properties, that can be used as a water-capturing device shown in the cuticle of the Namib desert beetle. 43 Xie group prepared a stable three-dimensional composite sponge with magnetic by situ coating of dopamine and followed by immersing in the mixture of magnetic Fe 3 O 4 nanoparticles and PDMS. The modified sponge exhibited high adsorption capacity for diverse organic solvents and can be easily recovered in oil–water mixture under magnetism. 44 However, the (super)wetting materials for effective oil–water separation are confined to some extents due to the inevitable rapid consumption of non-renewable and their high-cost. Additionally, the polluted separation materials are directly discarded or burnt, inevitably leading to the secondary pollution to the environment, although the oil pollution problem has been solved. Therefore, it is very important to develop eco-friendly and low-cost green materials for oil/water separation. In this work, environmentally friendly and abundant tissue paper with hydrophobicity is developed by coating of dopamine and followed by immersing in the mixed suspension of PDMS and SiO 2 nanoparticles. The as-prepared PDMS–SiO 2 /PDA/paper exhibits high porosity, strong water repellence, low water-adhesion force, and excellent mechanical strength. Moreover, the highly efficiency and large flux for treating multiple type oil–water mixtures was achieved, and the separation mechanism was investigated in detail.",
"discussion": "Results and discussion Surface morphology and chemistry The papers were fabricated by mussel-inspired surface coating technique, their micro-structures characterized as by SEM are shown in Fig. 2 , and the elemental weights measured by EDS from Fig. 2B are listed in Table 1 . The white pristine paper shows a three-dimensional structure consisting of microfibers with a diameter of 11.4 ± 0.3 μm. Only peaks of C and O are detected by EDS. After coating PDA for 12 h, the granules distribute loosely and randomly on the fibers, and the color of PDA/paper changes into dark brown. Three elements of C, O and N are simultaneously presented, and N accounts for 7.8 wt%. Upon being attached by PDMS and SiO 2 nanoparticles, PDMS–SiO 2 micro/nanoparticles overlapped densely around PDA particles, and the resultant PDMS–SiO 2 /PDA/paper becomes light. In addition, seen from Table 1 , new Si element appears and accounts for 43.2 wt%, while N can be neglected. It confirmed that PDMS–SiO 2 layer was successfully coated on PDA/paper. White PDMS–SiO 2 /paper exhibits a significantly lower roughness, and the elements of C, O and Si were also detected. The results indicated that PDA particles play important role in development of a micro/nanoscale hierarchical structure on the fiber surface. 44,45 In addition, after surface coating, the thickness increases, the porosity decreases, tensile strength and elongation at break of the papers scarcely change ( Table 2 ). Mussel-inspired surface modification technique is mild and facile. Fig. 2 (A) Photographs, (B and C) SEM images of the papers before and after mussel-inspired surface coating. Elemental weights of the samples characterized by EDS from SEM images in Fig. 2B Sample Elements (wt%) C O N Si Paper 56.9 43.1 — — PDA/paper 67.2 25.0 7.8 — PDMS–SiO 2 /PDA/paper 17.5 38.9 0.4 43.2 PDMS–SiO 2 /paper 30.7 41.0 — 28.3 Thickness, porosity, tensile strength and elongation at break of the papers before and after surface coating Sample Thickness (μm) Porosity (%) Tensile strength (MPa) Elongation at break (%) Paper 265.4 ± 3.4 87.4 ± 1.3 7.8 ± 0.6 23.8 ± 2.3 PDA/paper 287.4 ± 3.6 81.2 ± 1.7 8.4 ± 0.5 26.3 ± 1.0 PDMS–SiO 2 /PDA/paper 298.8 ± 5.1 77.0 ± 2.1 7.7 ± 0.8 27.5 ± 1.6 PDMS–SiO 2 /paper 293.4 ± 7.4 69.2 ± 3.5 8.1 ± 0.7 28.9 ± 2.7 Surface wettability The contact angles of the paper before and after surface modification are shown in Fig. 3A . The water contact angles in air of paper (a and e) and PDA/paper (b and f) are about 0°, and their underwater oil (soybean oil) contact angles are 145 ± 1.6 (i) and 151 ± 2.4° (j), thus suggesting a superhydrophilic and underwater oleophobic properties. Upon modified by PDMS–SiO 2 , tissue papers become hydrophobic (c and d). Compared with PDMS–SiO 2 /paper, PDMS–SiO 2 /PDA/paper is more hydrophobic with a water contact angle in air of ∼153° (g) and an underoil (trichloromethane) water contact angle of ∼144° (k). Furthermore, the bouncing behavior of water droplet on the hydrophobic surface was investigated. As shown in Fig. 3B , the water droplet rebounds on the superhydrophobic surface of PDMS–SiO 2 /PDA/paper without wetting or even residual. While, the water droplet sticks to the surface of PDMS–SiO 2 /paper and finally rest on the surface. The water-adhesion force of PDMS–SiO 2 /paper (229.3 μN) is obviously larger than that of PDMS–SiO 2 /PDA/paper (104.9 μN) ( Fig. 3C ). All the results illustrated that the satisfactory superhydrophobic modification of the paper was achieved through the comprehensive utilization of PDA and PDMS–SiO 2 , and liquids on the surface are in Cassie–Baxter state (low adhesive surface). This is because of a high surface roughness obtained by self-polymerization and deposition of dopamine as shown in Fig. 2 . Moreover, it is attributed to more PDMS brushes and SiO 2 nanoparticles being generated. 30 Fig. 3 (A) Digital pictures of the water droplets dyed by methylene blue on the papers (a to d), water contact angles in air (e to h), underwater oil (soybean oil) contact angles (i and g), underoil (trichloromethane) water contact angles (k and l) of the paper before and after modification. (B and C) Photographs of water droplets bouncing on PDMS–SiO 2 /PDA/paper and PDMS–SiO 2 /paper. (D) Water-adhesion forces on the sample surfaces. Absorption of liquid Owing to open three-dimensional structure of the papers before and after modification, they showed great potential for the selective adsorption of liquid from water/oil mixture. As shown in Fig. 4A , PDMS–SiO 2 /PDA/paper is able to adsorb trichloromethane (dyed with oil red O) immersing in water within 3 s. In addition, the adsorption capacity of the samples for various liquid was calculated and the results are shown in Fig. 4B . It is indicated that the adsorption capacity of water for paper is almost the same as that of PDA/paper, due to their similar micro-structure and hydrophilicity. Compared with PDMS–SiO 2 /paper, PDMS–SiO 2 /PDA/paper has a high absorption capacity for 1,2-dichloroethane, trichloromethane and soybean oil. Fig. 4 (A) Photographs of the underwater oil (trichloromethane) capture and collection process by PDMS–SiO 2 /PDA/paper. (B) Absorption capacity of the papers before and after surface coating for various liquids. Oil/water separation The separation performances of the paper before and after surface modification for different oil/water mixtures were investigated under gravity driving conditions. Upon dumping soybean oil/water into the apparatus, water quickly penetrates through the sample of paper due to it superhydrophilicity, while soybean oil is retained on the surface because of its under-water oleophobicity, as depicted in Fig. 5A . The corresponding water flux was more than 10 000 L m −2 h −1 , and the separation efficiency (SE) is 93.8 ± 1.4% ( Fig. 5C ). In addition, paper showed stable oil/water separation performance after 10 cycles of reuse. PDA/paper also can effectively separate soybean oil/water mixture. The separation treatment of immiscible heavy oil (carbon tetrachloride, trichloromethane, 1,2-dichloroethane)/water was similarly conducted by PDMS–SiO 2 /PDA/paper and PDMS–SiO 2 /paper. As shown in Fig. 5B , heavy oil penetrates through the sample due to its oleophilicity, while water is collected on the surface because of its under-oil hydrophobic properties. As summarized in Fig. 5D , compared with PDMS–SiO 2 /paper, the corresponding oil fluxes of PDMS–SiO 2 /PDA/paper are obviously larger, ascribing to higher porosity and under-oil hydrophobicity. The SE values of the samples for these heavy oil/water mixtures were found to be higher than 99%, demonstrating almost complete separation processes. More important, after 10 cycles, oil fluxes of PDMS–SiO 2 /PDA/paper and PDMS–SiO 2 /paper are similar to the initial values respectively, suggesting the oil/water separation behavior of the samples is fully reversible. And the surface morphology and chemical composition of the samples after 10 cycles of reuse hardly change as shown in Fig. 5F . The results indicated that all the papers before and after modification showed stable oil–water separation performance and chemical stability. Fig. 5 (A) Photograph of soybean oil/water separation using paper. (B) Photograph of water/trichloromethane separation using PDMS–SiO 2 /PDA/paper. (C) Water flux for soybean oil/water mixture. (D) Oil flux and separation efficiency for various oil/water mixtures. (E) The recycled separation flux of 1,2-dichloroethane/water mixture. (F) SEM images and elemental weights characterized by EDS of the samples after 10 cycles of reuse. Stability The significant feature of the materials for efficient separation oil/water mixture is the stability and durability in acidic, alkaline and salty. In this test, the as-prepared hydrophobic PDMS–SiO 2 /PDA/paper was first immersed in ethanol for 30 min, taken out and shaken in aqueous solutions at pH 1, 3, 7, 10, and 13, as well as aqueous solution of 2 M NaCl at 60 °C for continuous 7 days. Compared with the pristine PDMS–SiO 2 /PDA/paper, the tensile strength and elongation at break of the washed samples in various solution changed barely. It was found that the solution color changes from colorless to brown, as pH value increases from 1 to 13, and the washed sample turns from dark drown to faint yellow ( Fig. 6A ). The strongly alkaline solution leads PDA disassembly or destacking. 33,46 The underoil (trichloromethane) water contact angle of the sample after washing is more than 140° ( Fig. 6A ). The multiscale structures of the washed samples are also observed in Fig. 6C . Fig. 6D shows the ATR-FTIR spectra of the samples. Compared with the pristine paper, all the spectra of the washed PDMS–SiO 2 /PDA/paper exhibit the characteristic signals of Si–C bond (at 1257 and 780 cm −1 ), Si–O–Si bond (at 1070 cm −1 ), and C–H stretching vibration (at 2962 cm −1 ), 30 suggesting the good stability of PDMS–SiO 2 layer on the PDA modified paper substrate. Seen from Fig. 6E , after immersion in the solution of pH 13, the flux of the modified papers washed under various solutions did not change much. All the results indicated that PDMS–SiO 2 /PDA/paper exhibits good stability in acidic, alkaline and salty. Fig. 6 (A) Digital pictures of the water droplets dyed by methylene blue, (B) underoil (trichloromethane) water contact angles, (C) SEM images, (D) ATR-FTIR spectra, and (E) oil fluxes for separation of 1,2-dichloroethane/water mixture of PDMS–SiO 2 /PDA/paper after shaken in acidic (pH 1, pH 3), pure water (pH 7), alkaline (pH 10, pH 13) and salty (2 M NaCl) at 60 °C for 7 days. Insets in (A) corresponding photographs of the different aqueous solutions with PDMS–SiO 2 /PDA/paper."
} | 4,043 |
39905983 | PMC12018827 | pmc | 6,177 | {
"abstract": "Summary Lignin is a crucial component of the cell wall, providing mechanical support and protection against biotic and abiotic stresses. However, little is known about wheat lignin‐related mutants and their roles in pathogen defence. Here, we identified an ethyl methanesulfonate (EMS)‐derived Aegilops tauschii mutant named brown glume and internode 1 ( bgi1 ), which exhibits reddish‐brown pigmentation in various tissues, including internodes, spikes and glumes. Using map‐based cloning and single nucleotide polymorphism (SNP) analysis, we identified AET6Gv20438400 ( BGI1 ) as the leading candidate gene, encoding the TaCAD1 protein. The mutation occurred in the splice acceptor site of the first intron, resulting in a premature stop codon in BGI1 . We validated the function of BGI1 using loss‐of‐function EMS and gene editing knockout mutants, both of which displayed reddish‐brown pigmentation in lignified tissues. BGI1 knockout mutants exhibited reduced lignin content and shearing force relative to wild type, while BGI1 overexpression transgenic plants showed increased lignin content and enhanced disease resistance against common root rot and Fusarium crown rot. We confirmed that BGI1 exhibits CAD activity both in vitro and in vivo , playing an important role in lignin biosynthesis. BGI1 was highly expressed in the stem and spike, with its localisation observed in the cytoplasm. Transcriptome analysis revealed the regulatory networks associated with BGI1 . Finally, we demonstrated that BGI1 interacts with TaPYL‐1D, potentially involved in the abscisic acid signalling pathway. The identification and functional characterisation of BGI1 significantly advance our understanding of CAD proteins in lignin biosynthesis and plant defence against pathogen infection in wheat.",
"introduction": "Introduction Bread wheat ( Triticum aestivum L.) is a crucial staple crop worldwide, contributing about 20% of caloric and protein intake for the human population. However, wheat production is threatened by various factors, including diseases and lodging. Common root rot (CRR) caused by Bipolaris sorokiniana and Fusarium crown rot (FCR) caused by multiple Fusarium species are important diseases in many arid and semi‐arid cropping regions worldwide, such as Australia, the United States, Canada, China and South Africa (Bozoğlu et al ., 2022 ; Kazan and Gardiner, 2018 ). These two diseases are major concerns in regions with extensive wheat‐maize rotation and straw returning practices, as seen in China (Su et al ., 2021 ). In addition to diseases, lodging is also a significant limiting factor for wheat production, reducing grain yield and causing several knock‐on effects, including decreased grain quality and increased drying costs (Berry and Spink, 2012 ). Enhancing lignin content to improve lodging resistance is a crucial breeding objective (Zhang et al ., 2016 ). Lignin is a complex and heterogeneous aromatic polymer that forms the secondary cell wall together with cellulose and hemicellulose. Lignin plays an important role in mechanical support, water conductance and protection against biotic and abiotic stresses during plant growth and development (Barros et al ., 2015 ; Gallego‐Giraldo et al ., 2020 ). Lignin is gaining increasing attention due to its crucial role in enhancing both abiotic stress tolerance and pathogen resistance (Lee et al ., 2019 ; Rong et al ., 2016 ; Xu et al ., 2020 ). The synthesis of lignin involves the polymerisation of monolignols, which are produced through the phenylpropanoid pathway and subsequently undergo hydroxylation and methylation processes (Boerjan et al ., 2003 ; Ralph et al ., 2004 ). Polymerisation of the three main monolignols ( p ‐coumaryl, coniferyl and sinapyl alcohols) resulted in the formation of various monomers, namely p ‐hydroxyphenyl (H), guaiacyl (G) and syringyl (S), respectively (Vanholme et al ., 2010 ). The monomers undergo oxidation by peroxidases and/or laccases, resulting in the formation of monolignol radicals that combinatorially couple with each other and with the developing lignin polymer. Cinnamyl alcohol dehydrogenase (CAD) plays a crucial role in the lignin biosynthesis pathway. It is responsible for catalysing the nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of cinnamaldehydes to cinnamyl alcohol, the final step in monolignol biosynthesis before polymerisation in the cell wall (Goffner et al ., 1992 ; Vanholme et al ., 2010 ). Disruption of CAD activity significantly increases the incorporation of cinnamaldehyde into the lignin polymer (Liu et al ., 2021c ). Plants with impaired CAD function may exhibit delayed flowering, reduced plant height and a characteristic brownish‐red to tan pigmentation in the leaf midrib, particularly in C4 grasses (Chen et al ., 2012 ; Liu et al ., 2021c ; Trabucco et al ., 2013 ). Previous studies indicated that a high level of coniferyl aldehyde leads to the reddish‐brown colouration (Tsai et al ., 1998 ). Complete genome sequencing and annotation have enabled the determination of the number of CAD genes in various species, which has led to their classification into three classes (Barakat et al ., 2009 ). Class I CADs exhibit a highly conserved primary sequence structure across most species and are involved in lignin deposition in the secondary cell walls during plant growth and development (Park et al ., 2018 ). Class II CADs represent a more extensive and diverse group of CAD isoforms, known to be associated with stress resistance (Rong et al ., 2016 ). Class III CAD members may show redundancy with Class I CADs, yet their precise functions remain unclear (Peracchi et al ., 2024 ; Xu et al ., 2011 ). In Arabidopsis , although nine CAD genes have been identified in the published reference genome (Sibout et al ., 2005 ), only three ( AtCAD1 , AtCAD4 and AtCAD5 ) have been validated as the enzymes involved in monolignol biosynthesis (Rong et al ., 2016 ). Similarly, among the 12 CAD genes identified in the rice genome, OsCAD2 has been directly linked to lignin biosynthesis (Zhang et al ., 2006 ), while OsCAD7 plays a role in culm mechanical strength (Li et al ., 2009 ). In maize, ZmCAD2 ( GRMZM5G844562 ) is a member of the CAD family that plays a crucial role in lignification. Mutants exhibiting brown leaf midribs were first identified in ZmCAD2 , leading to their designation as brown midrib1 ( bm1 ) (Chen et al ., 2012 ). In sorghum, the bmr6 phenotype resulted from a mutation in the CAD gene, which is orthologous to the maize BM1 gene (Saballos et al ., 2009 ). The brown midrib trait is a valuable genetic characteristic that enhances the digestibility and quality of maize and sorghum, especially in livestock feed and bioenergy production. Recently, in silico analysis of the hexaploid wheat genome has revealed 47 high‐confidence TaCAD gene copies (Peracchi et al ., 2024 ). Among these TaCAD genes, TaCAD1 was speculated to be involved in lodging resistance (Chen et al ., 2021 ; Ma, 2010 ), and TaCAD12 was found to contribute to host resistance against sharp eyespot (Rong et al ., 2016 ). However, the functionality of these genes has not been experimentally verified in wheat. The challenges posed by the size and complexity of the bread wheat genome have historically impeded the identification and characterisation of TaCAD genes. Currently, there is a lack of data on lignin‐related CAD mutants or overexpression in wheat. This study addresses this gap by using a lignin‐deficient mutant called brown glume and internode 1 ( bgi1 ) identified from an EMS‐mutagenised library of the diploid Aegilops tauschii accession PI 511383. The bgi1 mutant plants exhibit a reddish‐brown pigmentation in various tissues, including internodes, spikes and glumes. Through map‐based cloning and single nucleotide polymorphism (SNP) analysis, we isolated the BGI1 gene, which encodes the TaCAD1 protein. We validated the function of the cloned candidate gene using loss‐of‐function EMS and gene editing knockout mutants. Furthermore, we investigated the regulatory mechanisms determining the role of BGI1 in lignin biosynthesis and demonstrated that BGI1 overexpression increases lignin content and enhances resistance against both CRR and FCR.",
"discussion": "Discussion The bgi1 mutant is defective in lignin biosynthesis \n CAD mutants have been previously characterised in several plant species, including bm1 in maize (Halpin et al ., 1998 ), bmr6 in sorghum (Li et al ., 2015 ; Scully et al ., 2016 ), gh2 in rice (Zhang et al ., 2006 ) and Bdcad1 in B. distachyon (Bouvier d'Yvoire et al ., 2013 ). While these studies identified CAD mutants, to our knowledge, no corresponding CAD mutants has been identified and characterised in wheat. Using map‐based cloning and SNP analysis, we identified the BGI1 gene encoding a CAD protein and found that bgi1 is a lignin‐deficient mutant, leading to a reddish‐brown colouration phenotype in lignified tissues. In maize and sorghum, the bm1 and bmr6 mutant plants exhibit reddish‐brown pigments in the stalk pith and leaf midrib (Halpin et al ., 1998 ; Saballos et al ., 2009 ). In rice, the gh2 mutant plants display pigments in the internode, hull and basal leaf sheath (Zhang et al ., 2006 ). Consistent with previous studies, our research identified that the bgi1 mutant plants show a reddish‐brown colouration in the internode, spike, spike rachilla, glume and lemma (Figure 1 ). All the above CAD mutants exhibit reddish‐brown colouration in lignified tissues, suggesting that these CADs play a conserved role in lignin biosynthesis across maize, sorghum, rice and wheat. However, phenotype differences were observed, such as the reddish‐brown colouration of the inner glumes in the bgi1 mutant, a trait not seen in other mutants. The difference in phenotypes could be attributed to two key factors. First, these CAD orthologous genes may have some degree of functional differentiation among different plant species. The maize BM1 (Halpin et al ., 1998 ) and sorghum BMR6 (Li et al ., 2015 ) genes were expressed strongly in leaves while rice GH2 (Zhang et al ., 2006 ), B. distachyon BdCAD1 (Bouvier d'Yvoire et al ., 2013 ) and wheat BGI1 genes were expressed slightly in the same tissue. Second, the type of mutation (missense or splice acceptor) may lead to different levels of reduction in CAD enzymatic activity. The gh2 mutant has an amino acid (G185D) change in the causal gene (Zhang et al ., 2006 ). In Brachypodium , the Bd4179 and Bd7591 mutant lines carry one (G192D) or two (G99V and S286F) amino acid changes in BdCAD1 (Bouvier d'Yvoire et al ., 2013 ). In contrast, the bgi1 mutant plants carry a mutation (G > A) in the splicing acceptor site of the first intron of BGI1 , leading to premature termination (Figure 2f ). These different types of mutations may contribute to the phenotypic differences observed across various plant species. The KO mutant plants of BGI1 exhibited reduced CAD activity and lower lignin content (Figures 5 and S11 ), which is consistent with observations in some monocotyledonous plants, such as maize (Halpin et al ., 1998 ; Xiong et al ., 2020 ), sorghum (Scully et al ., 2016 ), rice (Zhang et al ., 2006 ) and B. distachyon (Bouvier d'Yvoire et al ., 2013 ). However, the estimates of lignin concentration can vary greatly depending on the extraction method used. Some studies have observed reduced CAD activity without a corresponding decrease in lignin content (Baucher et al ., 1996 , 1999 ; Christiane Marque et al ., 1998 ). Overexpressing \n BGI1 \n results in increased lignin content and shearing force Numerous studies have documented CAD mutants, yet research on the phenotypic changes resulting from CAD overexpression in cereal crops remains relatively scarce. In Artemisia annua , overexpression of the AaCAD gene led to significantly higher lignin content in transgenics compared with WT plants (Ma et al ., 2018 ). Transgenic Arabidopsis plants overexpressing IbCAD1 exhibited increased lignin content in stems and roots, with a higher proportion of S lignin compared to G lignin (Kim and Huh, 2019 ). PpCAD2 overexpression transgenic tomato plants had a higher lignin content and CAD enzymatic activity in the leaf, stem and fruit pericarp tissues (Li et al ., 2019 ). Although none of these studies were conducted in cereal crops, these results support our observation that overexpression of BGI1 leads to increased lignin content (Figure S11 ). In addition to increasing lignin content, BGI1 overexpression significantly enhances stem strength, as evidenced by increased shearing force of stems (Figure S12 ). Recent correlation analyses have identified a significant positive relationship between TaCAD1 gene expression and stem strength (Chen et al ., 2021 ). Lodging is a common issue in wheat production, causing yield losses ranging from 10% to 80% (Easson et al ., 1993 ; Peng et al ., 2014 ). Lignin accumulation has been positively and significantly correlated with internode breaking strength and culm lodging resistance (Peng et al ., 2014 ). Thus, overexpression of BGI1 could be a potential strategy for improving lodging resistance in wheat. Interestingly, overexpression of PpCAD2 in tomato resulted in increased plant height, longer roots and larger stem diameter (Li et al ., 2019 ). Overexpression of IbCAD1 in Arabidopsis enhanced seed germination rate and increased tolerance to reactive oxygen species (Kim and Huh, 2019 ). Although the BGI1 overexpression transgenic plants did not exhibit any significant phenotypic changes (Figure S22 ), comprehensive agronomic and quality evaluations are necessary. Overexpression of \n BGI1 \n enhances pathogen resistance Yield losses caused by B. sorokiniana are often severe, ranging from 15% to 20%. Under favourable conditions of heat and drought, this disease can reduce wheat production by up to 70% and cause significantly seed quality deterioration (Sharma and Duveiller, 2007 ). FCR infection is estimated to cause a 35% yield loss in winter wheat in the Pacific Northwest region of the United States, with wheat seeds likely to become contaminated with fungal toxins (Smiley et al ., 2005 ; Su et al ., 2021 ). Additionally, there are only a limited number of genetic loci conferring resistance to CRR or FCR (Su et al ., 2021 ). In our study, transgenic plants overexpressing BGI1 exhibited increased resistance against both CRR and FCR infections (Figure 6a,b ). This enhanced resistance to pathogen infections in OE transgenic plants is linked to the increased lignin content. Lignin serves as a mechanical defence barrier and is known to be involved in plant defence against pathogen infections (Miedes et al ., 2014 ; Vance et al ., 1981 ). Lignification can form protective barriers against pathogen invasion, modify cell walls to resist pathogen‐released degrading enzymes, enhance the resistance of cell walls to toxins diffusion from pathogens to hosts, generate free radicals and toxic precursors and lignify to entrap pathogens (Bhuiyan et al ., 2009 ; Rong et al ., 2016 ). In Arabidopsis , genetic and functional analyses indicate that CAD‐C and CAD‐D not only serve as key enzymes in lignin biosynthesis but also play vital roles in plant defence against Pseudomonas syringae pv. tomato (Tronchet et al ., 2010 ). In wheat, increased lignin accumulation has been associated with increased resistance against FCR (Yang et al ., 2021 ). Likewise, wheat lines overexpressing TaCAD12 demonstrated significantly enhanced resistance to the fungus Rhizoctonia cerealis throughout all growth stages (Rong et al ., 2016 ). These results support our findings that overexpression of BGI1 leads to increased pathogen resistance. In Arabidopsis , cad‐C and cad‐D mutations reduced the expression of PR1 and PR5 genes after inoculation with Pseudomonas syringae pv. Tomato (Tronchet et al ., 2010 ). In wheat, transcriptional levels of PR10 and PR17 c were significantly elevated in the stems of TaCAD12 ‐overexpression wheat plants, contributing to increased resistance against R. cerealis (Rong et al ., 2016 ). However, our study did not observe changes in transcript levels of PR genes in BGI1 ‐overexpressing plants upon pathogen inoculation (Figure S15 ), suggesting that CAD overexpression may involve different mechanisms in plant defence. The precise molecular mechanisms underlying the pathogen resistance caused by BGI1 overexpression require further investigation. The interaction between BGI1 and TaPYL ‐ 1D may contribute to the process of lignification Previous studies have reported that the hormone ABA plays a crucial role in regulating plant secondary cell‐wall deposition and lignification (Brookbank et al ., 2021 ; Liu et al ., 2021a ). ABA signalling is mediated by intracellular PYL receptors, which bind to and inhibit PP2Cs, thereby releasing protein kinases SnRK2s from inhibition (Chen et al ., 2020 ; Park et al ., 2009 ). In muskmelon, exogenous ABA enhanced CAD activity and promoted the production of lignin monomers, lignin content and phenolic acids in fruit wounds (Wang et al ., 2024b ). In Kenaf ( Hibiscus cannabinus L.), ABA treatment induced a biphasic expression pattern of HcCAD2 , with significant induction at 6, 24 and 48 h (Choi et al ., 2016 ). Additional studies revealed that ABA increased the expressions of lignin biosynthesis genes, such as CAD , 4‐coumarate‐CoA ligase and cinnamate 4‐hydroxylase , as well as the activities of lignifying enzymes, thereby promoting lignin accumulation (Cheng et al ., 2013 ; Liu et al ., 2021b ; Xu et al ., 2020 ). These studies suggest a potential regulatory mechanism involving ABA, PYL receptors and CAD proteins in lignin biosynthesis. Our study identified a physical interaction between BGI1 and TaPYL‐1D using Y2H, LCI, BiFC and GST pull‐down assays (Figure 7d–g ). This finding aligns with GO annotations indicating enrichment in the ABA metabolic process (Figure S17b ), suggesting a regulatory role for BGI1 in ABA signalling. Our results provide a foundation for further investigation into the functional mechanisms of BGI1 and TaPYL‐1D in regulating lignin biosynthesis in wheat. In summary, we successfully isolated the bgi1 gene in wheat, which encodes the TaCAD1 protein. Functional validation through loss‐of‐function EMS and CRISPR mutations in BGI1 results in a reddish‐brown colouration of lignified tissues. Disruption of BGI1 significantly reduces lignin content without affecting plant development, potentially enhancing the use of wheat residues for biofuel production and other bio‐based products. Conversely, overexpression of BGI1 elevates lignin content and boosts pathogen resistance, which could lead to the development of wheat varieties with improved disease tolerance and lodging resistance. Specifically, increased disease resistance for CRR and FCR could reduce reliance on chemical control methods, contributing to more sustainable agricultural practices."
} | 4,838 |
33132890 | PMC7561669 | pmc | 6,179 | {
"abstract": "Existing mobile robots cannot complete some functions. To solve these problems, which include autonomous learning in path planning, the slow convergence of path planning, and planned paths that are not smooth, it is possible to utilize neural networks to enable to the robot to perceive the environment and perform feature extraction, which enables them to have a fitness of environment to state action function. By mapping the current state of these actions through Hierarchical Reinforcement Learning (HRL), the needs of mobile robots are met. It is possible to construct a path planning model for mobile robots based on neural networks and HRL. In this article, the proposed algorithm is compared with different algorithms in path planning. It underwent a performance evaluation to obtain an optimal learning algorithm system. The optimal algorithm system was tested in different environments and scenarios to obtain optimal learning conditions, thereby verifying the effectiveness of the proposed algorithm. Deep Deterministic Policy Gradient (DDPG), a path planning algorithm for mobile robots based on neural networks and hierarchical reinforcement learning, performed better in all aspects than other algorithms. Specifically, when compared with Double Deep Q-Learning (DDQN), DDPG has a shorter path planning time and a reduced number of path steps. When introducing an influence value, this algorithm shortens the convergence time by 91% compared with the Q-learning algorithm and improves the smoothness of the planned path by 79%. The algorithm has a good generalization effect in different scenarios. These results have significance for research on guiding, the precise positioning, and path planning of mobile robots.",
"conclusion": "Conclusions Through neural networks, the fitting from the environment to the state action function was realized by perceiving the environment and performing feature extraction. Through the enhancement function, the mapping of the current state to the action of the hierarchical reinforcement learning was satisfied, thereby enabling the robot to become more mobile. The two were organically combined to improve the performance of mobile robots during path planning. The mobile robot path planning algorithm based on neural networks and hierarchical reinforcement learning has better performance than other algorithms in all aspects. In addition, the proposed algorithm reduces the planning time, decreases the number of path steps, shortens the convergence time, and increases the smooth and efficient recognition and movement functions of the mobile robots. Although the performance of each algorithm has been analyzed as comprehensively as possible, the following aspects need to be improved in the future. First, it is impossible for the neural network learning method of the mobile robot's motion path planning to perform multiple “trial and error” processes in actual operations, which makes it difficult to apply the proposed algorithm. It is therefore necessary to implement the application on the physical platform before applying the algorithm to the actual robots. Second, the path planning only involves static scenarios. Whether the algorithm can show the same performance when encountering dynamic environmental changes is yet to be explored. The path planning capabilities of mobile robots were improved, laying a theoretical foundation for practical applications.",
"introduction": "Introduction Mobile robot autonomous navigation can be divided into three subsystems: information perception, behavior decision-making, and manipulation control. Path planning is the basis of mobile robot navigation and control (Ghosh et al., 2017 ; Orozco-Rosas et al., 2019 ). The goal of mobile robot path planning is to find a path from the current position to the target position. The path should be as short as possible, the smoothness of the path should meet the dynamics of the mobile robot, and the safety of the path should be collision-free (Han and Seo, 2017 ). Depending on how much information is known about the environment in the path planning process, path planning can be divided into global path planning and local path planning (Li and Chou, 2018 ). There are many methods of path planning. According to specific algorithms and strategies, path planning algorithms can be roughly divided into four types: template matching, artificial potential field, map construction, and artificial intelligence (Zhao et al., 2018 ). Each type of path planning algorithm has an optimal application scenario and limitations. The current path planning of mobile robots relies heavily on the surrounding environment. In addition to the limitations of traditional path planning, robots cannot complete their learning and judgment in complex environments, a bottleneck in the development of research in this field (Bakdi et al., 2017 ). It is therefore particularly important to develop a path planning method with low reliance on the environment, which can quickly adapt to the surrounding environment. The Deep Q-Learning Network (DQN) is a way of modeling the environment and calculating the collision energy function, which is the main cause of a loss in functionality (Ohnishi et al., 2019 ). To realize the path planning process, the neural network is trained to minimize the loss function through the gradient descent method. To enable better generalization ability in the neural network, various sample data are needed for learning and training, however, an over large data sample will increase the training time (Shen et al., 2019a ; Sung et al., 2020 ). Deep Reinforcement Learning (DRL), as an important machine learning method, has received more attention and there are increasing applications of it in robot path planning DRL (Arulkumaran et al., 2017 ). The agent obtains knowledge through the exploration of an environment and learns using a process of trial and error. The DRL method has obvious advantages in path planning and requires less prior information about the environment (Wulfmeier et al., 2017 ; Zheng and Liu, 2020 ). Unlike the supervised learning method, reinforcement learning does not require much sample data for training, like neural network methods, and acquires sample data during the training process. In recent years, scholars have focused on using new algorithms or fusion algorithms to improve the performance of mobile robots (Yan and Xu, 2018 ). Lei et al. found that adding the Q-Learning algorithm to the reinforcement learning path enhances the ability of robots to dynamically avoid obstacles and local planning in the environment (Lei et al., 2018 ; Liu et al., 2019 ). Wang et al. found that compared with Distributed DQN (DDQN) algorithm, the Tree Double Deep Network (TDDQN) has the advantages of fast convergence speed and low loss (Wang P. et al., 2020 ). By using a neural network to strengthen the learning path planning system, Wen et al. suggested that the mobile robot can be navigated to a target position without colliding with any obstacles and other mobile robots, and this method was successfully applied to the physical robot platform (Wen et al., 2020 ). Botteghi et al. introduced a reward function training strategy in the fusion algorithm, which not only outperformed the standard reward function in terms of convergence speed but also reduced the number of collisions by 36.9% of iteration steps (Shen et al., 2019b ; Botteghi et al., 2020 ). Therefore, the fusion algorithm has obvious advantages in path planning and algorithm performance. However, the path planning performance of current fusion algorithms is not outstanding. Taking into account the shortcomings of these research results, we designed a mobile robot path planning system based on neural networks and hierarchical reinforcement learning. Through neural networks, this system perceives the environment and performs feature extraction to realize the fitting from the environment to the state action function (Chen, 2018 ). The mapping of the current state to the action of the hierarchical reinforcement learning is satisfied through the enhancement function, thereby realizing the demand for mobile robots. Theoretically, the organic combination of the two can improve the performance of mobile robots in path planning. Therefore, in this study, the algorithm was embedded into a mobile robot, and the designed algorithm was verified by comparing it with other path planning algorithms in different environments and scenarios. The initial Q -value of the proposed algorithm sped up the convergence speed, redefined the number of states, as well as the direction of motion, and step length. The real-time performance of the mobile robot's path planning and smoothness was significantly improved, and could be used to guide robot movement, and improve algorithm mobility (Liu and Wang, 2019 ).",
"discussion": "Results and Discussions Experimental Results of Different Path Planning Algorithms of Mobile Robot Figure 7 shows the experimental results of the path planning of mobile robot under different algorithms. As shown in Figure 7 , under the same starting and ending conditions, all algorithms can effectively avoid obstacles. Comparing Figures 7A,B , it was found that in the traditional Q-Learning and A3C algorithms, the reinforcement learning algorithm effectively reduces the number of path steps. Comparing Figures 7A,C , it was found that the introduction of a neural network algorithm based on the traditional Q-Learning algorithm can greatly reduce the number of paths and achieve the same effect as the reinforcement learning algorithm. Comparing Figures 7C,D , it was found that the introduction of the force field based on the neural network has greatly accelerated the running speed of the algorithm, causing a significant reduction in the number of steps. Although the algorithm can effectively avoid obstacles, it has taken many useless paths. Therefore, the DDQN algorithm of Q value accumulation was added. As shown in Figure 7E , the algorithm can effectively utilize the neural network to learn and achieve the minimum number of steps. Compared to the DQN algorithm, the running speed of DDQN was improved and compared to the PDQN algorithm, the DDQN can find the optimal path. As shown in Figure 7F , a reinforcement learning algorithm was added based on the neural network. It was found that compared to the DDQN algorithm, it runs faster and has an optimal path. According to the above results, the fusion algorithm using a neural network and reinforcement learning has better performance in the path experiment. Figure 7 Experimental results of different path planning algorithms of mobile robot. Performance Evaluation of Different Path Planning Algorithms of Mobile Robot Figure 8A illustrates the path planning time of different algorithms under different path lengths. The results show that as the path length increases, the path planning time is also increasing, where the time required is proportional to the path length. As far as different algorithms are concerned, the traditional Q-Learning algorithm takes the longest time, with an average of 78.35 s. The PDQN takes the shortest time because the algorithm introduces a force field, causing the algorithm to be improved continuously. The DDPG algorithm based on neural networks and HRL marks the second position, which takes an average of 40.7 s and is 48.05% higher than the traditional algorithm, 31.01% higher than the DQN algorithm of the neural network, and 40.1% higher than the reinforcement algorithm. Figure 8 Performance evaluation of time and steps of different mobile robot path planning algorithms (QL algorithm represents the Q-Learning algorithm). Figure 8B illustrates the number of path steps of different algorithms at different iteration times. As the number of iterations increases, it does not affect the Q-Learning and A3C algorithms because these two algorithms do not have deep learning capabilities. With the increase in the number of iterations, in terms of other algorithms, the number of path steps continues to decrease under the same path. Of the different algorithms, the reinforcement learning algorithm is significantly better than the traditional Q-Learning algorithm, with a 20.56% improvement. Of the different neural network algorithms, the DDPG algorithm has the best performance, which has an average path step of 63 steps; compared to the DQN algorithm, it has an increase of 20.25%. When compared to the DDQN algorithm, the number of path steps is increased by 8.69%. According to the above results, the PDQN algorithm is more efficient under the same path conditions, as the learning continues, the fusion algorithm performs better in terms of path steps. Figure 9A illustrates the convergence time of different algorithms under different path steps. The results show that as the path steps continue to increase, the convergence time of each algorithm is continuously increasing. Compared to the Q-Learning and A3C algorithms, after adding reinforcement learning, the convergence time of robot path planning is increased by 13.54%; compared to the Q-Learning and DQN algorithms, after adding the neural network algorithm, the convergence time of robot path planning is increased by 33.85%, which is the most obvious improvement. Comparing different neural networks, it was found that the convergence time of the DDQN algorithm with increased Q -value is greatly improved, and the convergence time of path planning is improved by 94.44% compared with the previous Q-Learning algorithm. For the DDPG algorithm based on neural network and HRL, the convergence time of the algorithm under the unsynchronized number is 1.34 s on average, which is 55.52% faster than the optimal DDQN algorithm. Figure 9 Evaluation of convergence time and cumulative reward performance of different path planning algorithms of the mobile robot (QL algorithm represents the Q-Learning algorithm). Figure 9B illustrates the cumulative rewards of different algorithms under different path steps. Since the designed reward rules are more stringent, the reward results are all negative, but this does not affect the obtained results. As shown in Figure 9B , as the number of path steps continues to increase, the cumulative rewards continue to increase. For different algorithms, comparing the Q-Learning and A3C algorithms, the cumulative reward is significantly improved by 29.64%. Compared to the Q-Learning algorithm, the neural network DQN has increased significantly. Under the same neural network, it was found that the PDQN algorithm that introduces the force field has less cumulative rewards. The reason may be that the purpose of the algorithm is to enhance the running speed of the algorithm. The mechanism for rewards is not very complete; thus, the rewards are less. Among the neural network algorithms, the DDQN algorithm has the best cumulative reward. However, compared to the fusion algorithm DDPG, the performance of the DDQN algorithm is not very good. The cumulative reward of DDPG is increased by 41.5% compared to DDQN. According to the above results, it is concluded that under different path steps, the convergence time of the algorithm is the fusion algorithm; at the same time, the algorithm can also obtain the most rewards. Analysis of Performance Changes in Neural Network and HRL Algorithms Under Different Environmental Conditions To explore the impact of different environmental conditions on the performance of the algorithm, the performance of the DDPG algorithm was tested under different action sets, grid numbers, state sets, and force values. Under the premise of the same starting point and ending point, the average value of the algorithm was obtained after running 30 times. The results are shown in Table 1 . As shown in the table, the comparison between M1 and M2 indicates that when the action set is doubled, the convergence time of the algorithm will increase by 41%, and the smoothness of the planned path is also increased by 53%. Comparing M2 and M3, it is found that when the number of grids is increased three times, the convergence of the algorithm will be reduced by 69%, and the smoothness will be increased by 45%. Comparing M3 and M4, it was found that increasing the number of state sets will slow down the convergence speed of the algorithm, but by adjusting the direction of the action set, the right angles and corners in the path can be avoided, and the smoothness with which it navigates the planned path is increased by 18%. Comparing M4 and M5, it is found that the introduction of the force field will reduce the convergence time of the algorithm by 49%, which can increase the action step size, thereby adjusting the number of state sets and the direction of the action set. Therefore, when the action set is 4, the number of grids is 3, and the state set is 40 * 40 * 8, with the introduction of the force value, the algorithm can reduce the convergence time by 91% compared with the traditional Q-learning algorithm, and the smoothness of the path increased by 79%. Table 1 Effect of different environmental conditions on algorithm performance. Numbering Number of states Number of actions Action step Potential field/s Convergence time Convergence round Path length Total corner/rad M1 40*40 4 1 N0 1.9254 682.6 38.1 21.677 M2 40*40 8 1 N0 2.7139 629.7 32.7 10.210 M3 40*40 8 3 N0 0.8515 274.6 34.3 5.655 M4 40*40*8 4 3 N0 1.4259 340.1 32.8 4.616 M5 40*40 4 1 Yes 0.9848 559.8 38.0 21.834 M6 40*40*8 4 3 Yes 0.1735 155.3 32.1 4.555 Analysis of Changes in Paths Based on Neural Networks and HRL Under Different Scenario Conditions Figure 10 and Table 2 indicate the path changes and quantitative data of the algorithm under different scene conditions. As shown in Figure 10 , by comparing Figures 10A,B , it was found that at the same starting point and ending point, under the condition of different obstacles, the algorithm system can effectively avoid obstacles and design the optimal paths. In addition, the convergence time is maintained at about 0.15 s, the number of convergence rounds is maintained at 145, and the total rotation angle is 4.8 rad. By comparing Figures 10A,C , it was found that under different environments and different starting points and ending points, the system can still avoid collisions with obstacles, maintain a high convergence time, and design an optimal path. Simulation results show that the proposed path planning algorithm for mobile robots based on neural networks and HRL has a good generalization effect in different scenarios. Figure 10 Path changes of algorithms in different scenarios. Table 2 Statistical results of algorithm path changes under different scenario conditions. Scenes Position Convergence time/s Convergence round Path length Total corner/rad P1 (1, 39) 0.1615 144.5 32.1 4.869 P2 (1, 39) 0.1468 147.0 31.6 4.712 P3 (39, 39) 0.1724 147.4 31.8 4.641\n\nDiscussion The neural network DQN can perceive the environment and perform feature extraction to realize the fitting from the environment to the state action function. This has been mentioned in the literature. Qiao et al. ( 2018 ) proposed an adaptive DQN strategy and applied it to text recognition. These results showed that the DQN algorithm is significantly better than other algorithms, which also indicated the advantages of the DQN algorithm in image recognition (Qiao et al., 2018 ). Compared with the deep learning algorithm DQN, the DDQN algorithm is better than DQN in terms of value accuracy and strategy, which is also consistent with previous reports (Qu et al., 2020 ). The hierarchical reinforcement learning technology is utilized to achieve the mapping from state to action and meet the mobile needs of mobile robots. The data have also proven that the robot path planning method based on deep reinforcement learning is an effective end-to-end mobile robot path planning method, which has also been confirmed in a study by Wang B. et al. ( 2020 ). The above results illustrate the feasibility of the proposed method in the path planning of mobile robots. The DDPG algorithm was developed based on the DQN algorithm. The biggest improvement is that the action strategy of the DQN algorithm can only select actions in discrete action space, while the DDPG algorithm can select actions in continuous action space. The results show that the algorithm is significantly better than other algorithms in terms of operating efficiency. This is consistent with the results of Shen X. et al. ( 2019 ), in which it was found that when compared with the exponential moving average the effective variance of DDPG and average DDQN were reduced, which explained the efficient runtime of the algorithm further (Shen X. et al., 2019 ). The results also found that after reinforcement learning is added, the convergence time of robot path planning is increased by 13.54%. Low et al. used the flower pollination algorithm to properly initialize the Q -value, which could speed up the convergence of mobile robots (Low et al., 2019 ). The principle is similar to reinforcement learning, therefore, the research results here are also supported. The comparison between the Q-Learning and DQN algorithms found that the convergence time of robot path planning is increased by 33.85% after adding the neural network algorithm. Some scholars have improved the convergence performance of the model significantly by using two natural heuristic algorithms in unknown or partially known environments (Saraswathi et al., 2018 ). This natural heuristic algorithm is similar to the neural network structure, further proving the effectiveness of the proposed algorithm. In summary, the proposed DDQN algorithm has been proven to be applicable to image feature extraction, and the neural network algorithm has also been proven to effectively improve the performance and convergence of the algorithm. The data obtained are consistent with previous research. However, in terms of algorithm performance, the performance of mobile robot path planning based on neural networks and hierarchical reinforcement learning has been significantly improved. This algorithm can significantly reduce path planning time and improve smoothness, enabling mobile robots to move more conveniently and flexibility."
} | 5,583 |
31294192 | PMC6604955 | pmc | 6,180 | {
"abstract": "Worldwide, arable soils have been degraded through erosion and exhaustive cultivation, and substantial proportions of fertilizer nutrients are not taken up by crops. A central challenge in agriculture is to understand how soils and resident microbial communities can be managed to deliver nutrients to crops more efficiently with minimal losses to the environment. Throughout much of the twentieth century, intensive farming has caused substantial loss of organic matter and soil biological function. Today, more farmers recognize the importance of protecting soils and restoring organic matter through reduced tillage, diversified crop rotation, cover cropping, and increased organic amendments. Such management practices are expected to foster soil conditions more similar to those of undisturbed, native plant-soil systems by restoring soil biophysical integrity and re-establishing plant-microbe interactions that retain and recycle nutrients. Soil conditions which could contribute to desirable shifts in microbial metabolic processes include lower redox potentials, more diverse biogeochemical gradients, higher concentrations of labile carbon, and enrichment of carbon dioxide (CO 2 ) and hydrogen gas (H 2 ) in soil pores. This paper reviews recent literature on generalized and specific microbial processes that could become more operational once soils are no longer subjected to intensive tillage and organic matter depletion. These processes include heterotrophic assimilation of CO 2 ; utilization of H 2 as electron donor or reactant; and more diversified nitrogen uptake and dissimilation pathways. Despite knowledge of these processes occurring in laboratory studies, they have received little attention for their potential to affect nutrient and energy flows in soils. This paper explores how soil microbial processes could contribute to in situ nutrient retention, recycling, and crop uptake in agricultural soils managed for improved biological function.",
"conclusion": "6. Conclusions In this review we have attempted to relate management practices intended to mimic native soil conditions with insights from the literature on CO 2 assimilation, H 2 oxidation, alternative N transformation pathways, and enhanced fungal involvement in N cycling. We have the properties of undisturbed, native soils as management targets for plant-soil systems that could result in less nutrient loss. At the same time, we acknowledge that native soils are not capable of delivering needed amounts of nutrients on a sustained basis for agricultural production as we know it. In order for agricultural systems to be assisted by soil microbial processes, appropriate plant choices, organic amendments, and soil management practices will be needed to establish soil conditions that permit sustained activity of diverse microbial metabolisms. We also acknowledge that soils which have undergone organic matter depletion for long periods will not return quickly to the conditions once extant in native soils. Nevertheless, less disturbed soils are expected to become more spatially and temporally heterogeneous eventually over time. One of the approaches to assess the importance of specific microbial metabolisms in increasing nutrient cycling efficiency will be to couple next generation sequencing and metabolomics with biogeochemical process measurements applied to soils with well-characterized management histories and field records. Such studies will help to identify taxa that play critical roles in improving nutrient cycling function and relate these agricultural management practices. Deeper understanding of potential soil microbial metabolisms is needed so that agricultural soils can be managed in a more ecologically and environmentally sustainable manner. Gaining insights into conditions that lead to ecologically beneficial microbial metabolisms will help to align agricultural management practices with efficient nutrient cycling and lower environmental impact.",
"introduction": "1. Introduction Native soil ecosystems have been converted for agricultural use since the dawn of human civilization. During the past century, global food demands have intensified land conversion, as well as use of fertilizers, irrigation, and mechanization [1] . Modern agriculture is dominated by large-scale, continuously mono-cropped fields that have incurred significant losses of soil and organic matter and require increasing amounts of fertilizers [2] , [3] . Reliance on synthetic fertilizer has increased due to decoupling of crop and livestock production and less use of manures and legume rotations to restore soil fertility. On a worldwide basis, less than 50% of fertilizer nitrogen (N), regardless of source, is taken up by crops [4] . Nutrient imbalances are exacerbated as livestock production becomes more concentrated, resulting in further losses of unused reactive N to the environment [5] . While modern agriculture has helped address the daunting challenges of increased population and food demand, it has also led to deterioration of the soil's capacity to sustain plant and microbial biodiversity and perform ecosystem services [6] , [7] . Native soils contain accumulated organic matter from decades to centuries of successional vegetation and decomposed litter, as well as intricate root-microbial networks belowground. Conversion of native soils to agriculture destroys the biological linkages between roots, mycelial networks, and interacting microorganisms, thus rendering soils more vulnerable to erosion [8] . Continuous agriculture precludes most plant residues from being returned to the soil, and repeated tillage further depletes soil organic matter through physical disruption and oxidation [9] . Awareness is growing, however, that a sustainable food supply calls for reversing decades of soil erosion and organic matter loss. More farmers are attempting to achieve this by reducing tillage, rotating crops, cover cropping, and returning more organic amendments to soils [3] , [10] , [11] , [12] . Reduced- or no-tillage helps restore soil biophysical integrity and stabilizes microbial habitats to facilitate nutrient exchanges among microbes and between microbes and plants. Less disturbed soils may support development of lower soil oxidation-reduction potentials to enable microbial metabolic diversification. Crop rotations and cover cropping introduce a wider variety of organic compounds through greater root densities. Such management practices could foster adaptive microbial diversity in soils for better nutrient reutilization and fewer losses to the environment [13] . This paper highlights beneficial microbial metabolisms that could become more operational once soils are no longer subjected to intensive tillage and organic matter depletion. It describes how management-induced soil conditions (i.e., improved physical structure, higher organic matter content) could promote such microbial processes as heterotrophic CO 2 consumption, H 2 utilization, and diversified N respiratory pathways in soils ( Figure 1 ). The rationale for this review is that biologically based agricultural management is expected to improve soil microbial habitat and increase microbial growth and diversity. When promoted in agricultural soils through management, these microbial processes could speed soil organic carbon (C) accretion, increase nutrient reuse, and reduce N losses to the environment. Figure 1. Schematic of agricultural management practices that aim to re-establish more native soil properties and create habitat conditions conducive to the microbial metabolisms highlighted in this review: heterotrophic CO 2 assimilation, H 2 oxidation, dissimilatory nitrate reduction to ammonium (DNRA), non-denitrifier N 2 O reduction, and fungal NO 3 − uptake. Respective colors of text, connecting arrows, and lines are blue (for physical structure improvement); green (for increased plant inputs); and red (for more diverse organic inputs)."
} | 1,988 |
34150503 | PMC8193244 | pmc | 6,183 | {
"abstract": "Synthetic biology approaches for the synthesis of value-based products provide interesting and potentially fruitful possibilities for generating a wide variety of useful compounds and biofuels. However, industrial production is hampered by the costs associated with the need to supplement large microbial cultures with expensive but necessary co-inducer compounds and antibiotics that are required for up-regulating synthetic gene expression and maintaining plasmid-borne synthetic genes, respectively. To address these issues, a metabolism-based plasmid addiction system, which relies on lipopolysaccharide biosynthesis and maintenance of cellular redox balance for 1-butanol production; and utilizes an active constitutive promoter, was developed in Escherichia coli . Expression of the plasmid is absolutely required for cell viability and 1-butanol production. This system abrogates the need for expensive antibiotics and co-inducer molecules so that plasmid-borne synthetic genes may be expressed at high levels in a cost-effective manner. To illustrate these principles, high level and sustained production of 1-butanol by E. coli was demonstrated under different growth conditions and in semi-continuous batch cultures, in the absence of antibiotics and co-inducer molecules.",
"conclusion": "5 Conclusion Competitive yields of 1-butanol were produced without the requirement of antibiotic or co-inducer supplementation over a time period of 9 days. Furthermore, the PAS system described here is versatile, being independent of carbon source or growth mode, as compared to other systems. Finally, a PAS-based 1-butanol production strain should lend itself well to industrial-scale, low-cost production, particularly with additional strain modifications. For example, it was previously demonstrated that 1-butanol production in E. coli could reach levels up to 30 g/L by over-expression of the formate dehydrogenase gene and continuous removal of 1-butanol by gas stripping from a pH controlled batch fermentor ( Shen et al., 2011 ). In addition, over-expression of the pyruvate dehydrogenase complex provided increased levels of NADH and markedly improved 1-butanol production in E. coli ( Bond-Watts et al., 2011 ). Based on our initial experiments, we might realistically expect that a similarly modified strain RKE09 might continuously produce high levels of 1-butanol, however without the need for co-inducers or antibiotics. The major conclusion from this study, however, is that plasmid-addicted strains containing constitutive promoters will be highly useful for synthetic biology approaches for low-cost industrial synthesis of bioproducts and biofuels.",
"introduction": "1 Introduction The advent and use of synthetic biology approaches has allowed the scientific community to engineer microorganisms for the production of a variety of value-based products, such as human insulin, proteases, and antibiotics ( Chance and Frank, 1993 , Adrio and Demain, 2014 , Thykaer and Nielsen, 2003 ). Recently, the microbial production of biofuels, such as 1-butanol, has attracted great interest and gained momentum due to several environmental, economic and political factors. The chemical properties of 1-butanol, its high energy density and low hygroscopicity, and its compatibility with the current infrastructure, make it an attractive candidate for a transportation fuel, compared to ethanol ( Peralta-Yahya and Keasling, 2010 ). Clostridium acetobutylicum , a Gram-positive strict anaerobic bacterium, has historically been used for the microbial production of 1-butanol, as well as acetone and ethanol. The genes responsible for 1-butanol production in C. acetobutylicum were identified ( Boynton et al., 1996 , Fontaine et al., 2002 ) and heterologous expression of these genes has been accomplished in several different microorganisms, such as Bacillus subtilis ( Nielsen et al., 2009 ), Lactobacillus brevis ( Berezina et al., 2010 ), Pseudomonas putida ( Nielsen et al., 2009 ), Saccharomyces cerevisiae ( Steen et al., 2008 ), and Escherichia coli ( Atsumi et al., 2008 , Shen et al., 2011 , Bond-Watts et al., 2011 , Gulevich et al., 2012 ) with particular interest in E. coli due to its proven industrial usage and associated extensive genetic and biochemical knowledge. Early constructs resulted in low yields of 1-butanol produced by wild-type organisms ( Atsumi et al., 2008 ). To overcome this, multiple metabolic and genetic factors were altered in efforts to increase 1-butanol production. For example, with E. coli changes in the growth conditions or medium, the use of plasmids or promoters for expression of the 1-butanol genes, the elimination of competitive pathways, and the use of homologous non-clostridial genes have all played a significant role in raising the level of 1-butanol production to impressive levels ( Shen et al., 2011 ). As opposed to chromosomal expression, plasmid-based systems are often used for up-regulated heterologous expression of synthetic genes in a non-native organism; this is the case for genes that are required for 1-butanol production. The use of a plasmid-based system has many advantages, as it allows for (1) an increase in enzyme pools by gene dosage, (2) control of gene expression if an appropriate promoter and co-inducer are chosen, and (3) rapid construction of different combinations of genes due to facile manipulation of plasmids, as opposed to insertion of genes on the chromosome. However, if one chooses to scale-up to industrial production levels, there are two major drawbacks with this laboratory bench method. The first is the combined cost associated with supplementing cultures with a co-inducer to induce gene expression and the use of antibiotics to maintain plasmid stability. Secondly, large scale-ups invite potential ecological issues associated with the usage of large amounts of antibiotics, such as the rise in antibiotic resistant bacterial strains. To overcome reliance on antibiotics, the metabolism-based plasmid addiction system (PAS) was devised ( Voss and Steinbuchel, 2006 ). Essentially, the PAS relies on the strict natural selection of plasmid-containing cells, due to the expression of a plasmid-encoded gene(s) that is required for the viability of the bacterium ( Kroll et al., 2010 ). Therefore, cells that maintain a plasmid containing the essential gene(s) and a suite of value-based product gene(s) are viable and able to produce the desired product. There are a few examples of the use of metabolism-based plasmid addicted systems for value-based product formation. The first example used Ralstonia eutropha strain H16. Plasmid expression of the essential 2-keto-3-deoxy-6-phosphogluconate (KDPG)-aldolase gene coupled with a cyanophycin synthetase gene, resulted in cyanophycin production with either fructose or gluconate as carbon source ( Voss and Steinbuchel, 2006 ). A second example was cyanophycin production in E. coli ( Kroll et al., 2011 ). Like the first example, plasmid addicted value-based product formation was medium- and carbon-source dependent. Another example was the plasmid-based expression of a synthetic 1-butanol operon in an E. coli mutant strain that restored anaerobic growth; as a consequence, 1-butanol was produced ( Shen et al., 2011 ). However, in this instance, expression of the plasmid-based operon was dependent on co-inducer, isopropyl β- d -1-thiogalactopyranoside (IPTG), addition. Recently, the production of ethanol was accomplished in E. coli and, similarly to other examples, plasmid addiction was carbon source-dependent ( Wong et al., 2014 ). To date, all recently employed PAS systems negate the requirement of antibiotics for plasmid stability. However, each of these PAS systems relies on specific constraints; for example, the use of a specific carbon source or medium-specific growth condition, as well as the need for co-inducer supplementation for up-regulated gene expression. There is one exception that does not rely on any constraints; however, in this example the product titer was less than that of the control strain ( Kroll et al., 2009 ). The aforementioned constraints limit the flexibility for industrial scale production. Addressing these issues, we now report the development of a plasmid addicted 1-butanol production system in E. coli that negates the need for expensive co-inducers and antibiotics, and is not limited by medium, carbon source, or growth condition. Without the constraints usually associated with metabolism-based plasmid addicted value-based product synthesis, this system produced significant yields of 1-butanol using a test strain of E. coli during semi-continuous batch culture.",
"discussion": "4 Discussion The ability to use microorganisms as biological factories for the production of value-based products is becoming commonplace due to (1) increased metabolic knowledge of selected host microorganisms, thus allowing for the manipulation of native biochemical pathways and (2) the use of synthetic biology principles whereby key genes encoding for desired foreign biochemical pathways may be used for synthesis of desired value-based products. In order to engineer microbes to serve as metabolic factories for industrial scale production of value-based compounds the selected system must be easy to manipulate and the costs associated with growing the organism for maximal product formation must be low. In this study we have developed a metabolism-based plasmid addiction system in E. coli for the production of a model bioproduct, 1-butanol, which retains the benefits of a plasmid-based system but without the cost associated with co-inducers to up-regulate gene expression or antibiotics to maintain plasmid stability. Moreover, the strategy we have developed abrogates environmental issues associated with large-scale usage of antibiotics. The plasmid addiction strategy developed here involves the expression of the essential plasmid-borne lptB gene, which is co-expressed with genes of a synthetic 1-butanol pathway, thus ensuring that microbial growth and cell viability involves synthesis of the desired product, 1-butanol in this case, without the need for antibiotics to maintain plasmid stability. In addition, when anaerobic growth was employed, the production of 1-butanol served as a second essential system for cell viability, thus increasing the stringency for plasmid addiction. It is important to note that our system employs a promoter that allows for constitutive expression of desired genes, such that expensive cofactors such as IPTG are unnecessary. As a result, under small-scale semi-aerobic conditions, the initial plasmid-addicted strain produced 56 mg/L of 1-butanol without the use of co-inducer and antibiotics. Further development of this strain, by inactivation of competing fermentative pathways, resulted in the production of 2.3 g/L under small-scale conditions, again without the requirement of co-inducers or antibiotics. Further extensive studies were performed under larger-scale growth conditions, with supplementation of growth media with glycerol or glucose under aerobic, anaerobic, semi-aerobic, and high cell density conditions. Using strain RKE09, a maximum titer of 4.7 g/L was produced under aerobic growth with 2% glycerol. Under anaerobic conditions, the 1-butanol titer was highest with glucose supplementation as opposed to glycerol addition, with a titer of 4.1 g/L of 1-butanol (yield: 0.15 g butanol/g of glucose utilized, and the productivity was at 0.044 g butanol produced/L/h). In addition, larger-scale cultures did not require the presence of antibiotics to maintain plasmid stability, with high levels of 1-butanol obtained in the absence of antibiotics. These studies with larger scale cultures confirmed initial results with the small culture studies and verified the stability of our PAS 1-butanol producing strain, regardless of culture size or growth mode. Finally, strain RKE09 was able to stably produce high levels of 1-butanol over a time period of over 200 h during semi-continuous growth with no hint of any instability."
} | 3,022 |
27303415 | PMC4880586 | pmc | 6,184 | {
"abstract": "Brachypodium distachyon ( Brachypodium ) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl: p -hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p -coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. The availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.",
"conclusion": "Conclusion This study identified considerable phenotypic diversity for a variety of biomass-related traits in a genotypically diverse set of seven Brachypodium inbred lines. The phenotypic differences between lines for a given trait are significantly large enough to warrant follow-up genetic studies of related RILs in order to identify the underlying genetic determinants. For example, a Bd21 × Bd3-1 RIL population was successfully employed to fine-map a Barley Stripe Mosaic Virus resistance gene (e.g., Cui et al., 2012 ). We have analyzed biomass digestibility in a Bd21 × Bd2-3 RIL population; our preliminary findings suggest that a few genetic loci underlie the majority of observed digestibility differences and that transgressive segregation may be occurring. Although multivariate analysis of the data from this study identified correlations between growth characteristics, cell wall compositions, and biomass digestibility, further analyses will be required to identify cause and effect relationships. Moreover, given that the number of lines analyzed were limited, it may be worth revisiting some of these traits using a larger number of lines so as to increase statistical power. This may uncover even larger phenotypic differences as well as confirm or rule out marginal or unexpected correlations.",
"introduction": "Introduction Brachypodium distachyon ( Brachypodium ) is an annual grass species native to southern Europe, northern Africa, the Middle East, and southwestern Asia. As a member of the grass subfamily Pooideae, Brachypodium is related to Triticum aestivum (wheat) and other temperate cereals as well as most of the forage grass species, making it a useful model for a wide range of biological aspects of cool season grass biology ( Draper et al., 2001 ; Mur et al., 2011 ; Barrero et al., 2012 ; Cui et al., 2012 ; Lee M.Y. et al., 2012 ; Figueroa et al., 2015 ; Zhong et al., 2015 ). The cell wall composition, growth architecture, and flowering time regulation of Brachypodium are similar to those of other grass species, making it a useful model for biomass improvement of dedicated bioenergy grass species such as switchgrass and Miscanthus ( Gomez et al., 2008 ; Vogel, 2008 ; Lee S.J. et al., 2012 ; Rancour et al., 2012 ). Brachypodium has a diploid genome that is one of the smallest and least repetitive of any grass species, which allowed for the rapid generation of a high-quality reference genome sequence for inbred line Bd21 ( International Brachypodium Initiative, 2010 ), followed by additional genome sequences from several genotypically diverse Brachypodium genotypes ( Vogel et al., 2009 ; Gordon et al., 2014 ). The genome sequences and germplasm resources for Brachypodium coupled with its simple growth requirements and inbred nature, make this species attractive for studies of the molecular basis of natural variation. Multiple studies have shown that Brachypodium exhibits extensive natural variation with respect to photoperiod responsiveness, vernalization requirements, and flowering time, with accessions broadly falling into winter and spring annual types ( Schwartz et al., 2010 ; Ream et al., 2014 ; Woods et al., 2014a , b ). Luo et al. (2011) surveyed 57 natural populations of Brachypodium for drought tolerance, and found significant phenotypic diversity based on principal component (PC) analyses of chlorophyll fluorescence and leaf water content under drought stress. Tyler et al. (2014) also identified considerable phenotypic diversity within a large collection of inbred Brachypodium lines, focusing on bioenergy-relevant traits including plant height, growth habit, stem density, and cell wall composition as inferred by near infrared spectroscopy (NIR) and comprehensive microarray polymer profiling (CoMPP). Although the Tyler et al. (2014) study identified significant Brachypodium natural variation with respect to hemicellulose and pectin compositions, the analyses were semi-quantitative and did not explore lignin composition or biomass recalcitrance, two traits centrally important to developing bioenergy crops for conversion to liquid biofuels. Rancour et al. (2012) performed a detailed quantitative analysis of Brachypodium cell wall composition including that of lignin in different tissue types throughout development, but did not assess phenotypic diversity or biomass recalcitrance. Therefore, for this study we chose seven inbred lines that were previously found to have a high level of genotypic diversity ( Vogel et al., 2009 ), and phenotypically assessed all of the major secondary cell wall components as well as possible relationships to biomass recalcitrance. The chosen lines are particularly useful in that their genome sequences are publicly available, as are recombinant inbred line (RIL) populations for crosses between many of them ( Gordon et al., 2014 ; Garvin, 2015 ). These findings and resources will facilitate and accelerate discovery of the underlying mechanisms controlling yield, biomass composition, and recalcitrance to conversion to biofuels.",
"discussion": "Discussion Grass vegetative biomass holds considerable potential as a feedstock for the generation of liquid biofuels owing to its high latent sugar content of approximately two thirds polysaccharides by dw ( Pauly and Keegstra, 2008 ) and widespread availability, with hundreds of millions of tons available annually in the U.S. alone ( U.S. Department of Energy, 2011 ). B. distachyon ( Brachypodium ) has emerged as a tractable model for studying a variety of traits ( Brkljacic et al., 2011 ). In this study, we quantified and compared cell wall composition differences between a set of seven Brachypodium inbred lines previously found to have a high level of genotypic diversity. We found considerable phenotypic variation, suggesting that studies of related RILs can identify underlying genetic pathways differences, the knowledge of which can be used to improve grasses for use in generating biofuels. This study builds upon work by Tyler et al. (2014) , who semi-quantitatively assessed polysaccharides composition but not lignin or biomass recalcitrance of a collection of Brachypodium lines. Our analyses identified strong positive correlations between ABSL lignin amounts, lignin G unit amounts and plant heights ( Table 4 ). It may be that taller stems accumulate more cell-wall-strengthening lignin richer in G units to facilitate crosslinking in response to greater bending and torsional stresses. In addition, higher solute pressures in xylem and phloem may be associated with the taller stems, which may induce lignin accumulation to strengthen the vasculature. Dramatic differences between the inbred lines were also identified with respect to lignin S unit content and S:G ratios ( Figures 3A–C ). Studies by others of Brachypodium and Arabidopsis enhanced saccharification mutants identified correlations between reduced S:G ratios and improved biomass digestibility ( Van Acker et al., 2013 ; Marriott et al., 2014 ; Silveira et al., 2015 ). However, our statistical analyses identified only a few correlations between lignin composition and biomass digestibility when considering all five digestibility treatments employed ( Table 5 ; Supplementary Table S1 ; Figure 4 ). One possible reason why there was not a clearer observed relationship between lignin and digestibility is that it may have been masked by significant effects of other cell wall components such as hemicelluloses, AGPs, arabinogalactans (AGs), extensins, and/or pectins. Consistent with this hypothesis, a study of a maize RIL population identified several genetic determinants for digestibility, none of which were associated with lignin ( Penning et al., 2014 ). Taken together, these results suggest that studies of the appropriate Brachypodium RILs could identify not only allelic variants controlling flux through the monolignol biosynthetic pathway but also determinants unrelated to lignin that underlie the observed biomass digestibility differences. Significant phenotypic variation was also observed in the quantified total amounts of neutral sugars derived from cell wall polysaccharides and other sugar-containing wall components ( Table 1 ). For example, Bd18-1 had 16% more xylose and 25% more arabinose in senesced stems plus leaf sheaths than Bd21. Most of the xylose and a portion of the arabinose in Brachypodium and other grasses is present in the hemicellulose glucuronoarabinoxylan (GAX; Carpita and Gibeaut, 1993 ; McCann and Carpita, 2008 ; Vogel, 2008 ; Scheller and Ulvskov, 2010 ). Thus it seems likely that these inbred lines vary in their relative amounts of GAX. Arabinose is also present in AGPs, AGs, extensins, and pectins ( Carpita, 1996 ; Seifert and Roberts, 2007 ). The relative amounts of these cell wall components in Brachypodium remain to be quantitatively determined, although Tyler et al. (2014) did detect relatively stronger signals with the AGP-specific JIM13 monoclonal antibody, compared to various pectin-specific antibodies, in Brachypodium CDTA- and NaOH-extracted cell wall materials. Our results also revealed that the amounts of galactose and rhamnose, which are predominantly found in pectins and AGPs ( Carpita, 1996 ; Seifert and Roberts, 2007 ; Caffall and Mohnen, 2009 ), varied significantly between inbred lines ( Table 1 ). Tyler et al. (2014) identified similar significant differences in pectin composition between Brachypodium inbred lines by using a CoMPP technique, employing five antibodies to detect epitopes of the pectin homogalacturonan (HG). Their study identified Bd21-3 and Bd21 as having the largest and smallest relative amounts of HG, with Bd3-1, Bd30-1, and Bd1-1 falling in between. Those relative differences in HG levels matched well with the relative differences in galactose amounts our analyses uncovered between the same inbred lines ( Table 1 ), thus providing cross-validation between the two studies. Our results showed that galactose levels (likely from pectins and AGPs) along with xylose and arabinose levels (xylose predominantly from hemicelluloses, and arabinose mostly from hemicelluloses, AGPs, extensins, and pectins) were strongly and positively correlated with biomass accumulation ( Table 4 ). Considerable evidence in dicotyledonous plants points toward pectin composition and structure, including differences in pectin methylesterification, influencing plant morphogenesis and growth ( Palin and Geitmann, 2012 ; Kim et al., 2015 ). AGPs also play essential roles in growth and development ( Tan et al., 2012 ). Pectins and AGPs have been studied relatively little in monocots, including grasses ( Xiao and Anderson, 2013 ). Therefore, these Brachypodium inbred lines could be useful tools for related studies. Besides the aforementioned differences in structural polysaccharide levels between the inbred lines, substantial differences in free glucose, sucrose, and starch amounts were identified ( Table 2 ). For example, free glucose amounts varied by as much as 10.75-fold in senesced stem plus leaf sheath tissues. Intriguingly, the relatively high glucose and sucrose amounts in Bd2-3 senesced stems plus leaf sheaths largely disappeared when plants were vernalized for 14 days, dropping to the amounts found in unvernalized Bd21 and Bd30-1 ( Figure 6 ). Taken together, these findings suggest there are likely genetic differences in how these lines partition and store carbohydrates. Slewinski (2012) pointed out the importance of understanding carbon sink-source dynamics in order to maximize grass crop yields and improve yield stability under stress conditions. Studies employing Brachypodium could be informative in this regard."
} | 3,553 |
29463788 | PMC5820350 | pmc | 6,188 | {
"abstract": "A transition toward sustainable bio-based chemical production is important for green growth. However, productivity and yield frequently decrease as large-scale microbial fermentation progresses, commonly ascribed to phenotypic variation. Yet, given the high metabolic burden and toxicities, evolutionary processes may also constrain bio-based production. We experimentally simulate large-scale fermentation with mevalonic acid-producing Escherichia coli . By tracking growth rate and production, we uncover how populations fully sacrifice production to gain fitness within 70 generations. Using ultra-deep (>1000×) time-lapse sequencing of the pathway populations, we identify multiple recurring intra-pathway genetic error modes. This genetic heterogeneity is only detected using deep-sequencing and new population-level bioinformatics, suggesting that the problem is underestimated. A quantitative model explains the population dynamics based on enrichment of spontaneous mutant cells. We validate our model by tuning production load and escape rate of the production host and apply multiple orthogonal strategies for postponing genetically driven production declines.",
"introduction": "Introduction Bio-based production of chemicals and fuels is important to develop a more sustainable society. However, it remains difficult to scale-up many processes that rely on engineered organisms to produce industrially relevant quantities of bio-compounds, which frequently require 100 m 3 fermentation volumes. Indeed, a lack of robustness of synthetic production strains is considered a main challenge for implementing large-scale bioprocesses 1 , 2 . Furthermore, despite advantages such as higher volumetric productivity, the industrial implementation of continuous fermentation is often limited by appearance of non-producer cells 3 – 5 . Indeed, declining productivity constrains the economic feasibility of most fermentation reactions to shorter fed-batch operations 6 , ultimately limiting our societal transition toward bio-based chemical and fuel production. Poor performance of bio-based processes is speculated to arise from phenotypic cell-to-cell variation rather than single-nucleotide polymorphisms (SNPs) 7 , 8 . Suboptimal physical reactor conditions such as limited aeration and stochastic gene expression are thought to underlie population heterogeneities 9 – 11 . As such, subpopulations have been observed to temporarily cease production, then resume production at an unpredictable time 12 . In addition, the high-level cellular biosynthetic activity required for economically viable bioprocesses might reduce the fitness of producer cells enough to select for non-producing mutant cells during industrially relevant timescales. Such genetic heterogeneity would be more detrimental than temporal phenotypic variations, as genetic heterogeneity results in the irreversible loss of production from a subpopulation in the fermentation tank. The fitness cost of biosynthesis is pathway-specific and arises from metabolic loads such as enzyme synthesis, DNA synthesis, protein misfolding, and drains on endogenous metabolites (required for glycolysis and redox power), but it can also result from the accumulation of toxic intermediates and by-products 13 – 18 . We employ the term “production load” to the sum of these effects, which present a selective disadvantage for productive cells in direct competition with non-productive cells. The fitness of a production organism can be improved in a variety of ways, including rational engineering 19 , adaptive laboratory evolution 20 , 21 , functional metagenomics 22 , and fermentation optimization 23 , 24 . Despite recent progress, production organisms still retain a fitness cost that cannot be eliminated that is directly linked to the burden of non-natural biosynthetic productivity. Accordingly, production cells may be selected against in competition with more fit non-producing cells. However, the extent to which such evolutionary processes limit fermentation output remains unclear and depends on eventual population size, production load, and the number of cell divisions required to reach industrial fermentation scales. Generating the fermentation population inside an industrially sized 200 m 3 fed-batch bioreactor involves a gradual scale-up from a master cell bank aliquot and requires approximately 60–80 cell generations to reach population sizes of approximately 10 20 cells. Such timescales and population sizes could allow for both the generation and selection of non-producing organisms and might allow these organisms to reach substantial densities in the final fermentation population. One mechanism that led to non-producing cells in early, engineered bioprocesses is the loss of plasmids that encode components of the biosynthetic pathway. Strategies have been developed to limit the loss of plasmid-borne pathway cassettes, including punishing mis-segregation using plasmid-encoded selection genes, toxin-antitoxin systems, and chromosomal integration of the pathway genes 25 – 27 . However, maintenance of the biosynthetic pathway cassette does not preclude the accumulation of genetic errors targeting pathway genes or central metabolic host genes in trans , which leads to a loss of biosynthetic activity and potentially improved fitness. Indeed, limiting the mutation rate in Escherichia coli by deleting error-prone DNA polymerases and chromosomal insertion sequences (ISs) has led to higher end-point l -threonine productivity and overexpressed recombinant protein titer 28 , 29 . Such reports suggest that genetic heterogeneity resulting from processes other than gene loss might play a key role in limiting fermentation productivity. However, the actual mechanism and population-level dynamics of such genetically driven production disruption remains poorly understood, preventing the establishment of a framework for explaining and addressing such production failure modes. In this study, we investigate the phenotypic and genotypic dynamics of E . coli strains engineered to produce mevalonic acid over timescales relevant to industrial-scale fermentations. Mevalonic acid is a precursor to the important secondary metabolite class of isoprenoids, acting as a chemical building block for colorants, medicines, flavors, fuels, and fragrances 30 . Using ultra-deep, time-lapse sequencing of the fermentation populations, we resolve diverse, previously difficult-to-decipher, and non-canonical IS transposition events that limit production.",
"discussion": "Discussion Bio-based production is a central contributor to the transition of our society toward a greener and more sustainable future. However, large-scale bioprocesses are hampered by high yield and productivity requirements, and many new processes cannot be made commercially viable due to declining performance at the scale-up step. Prior work has focused on the phenotypic variance that can contribute to the reduced performance of bio-based processes 50 . While IS elements have been shown to disrupt production phenotypes, the role of evolution and genetic mechanisms in production decline is not well characterized. In this study, we experimentally simulated the timescales and population sizes of large-scale bioprocesses in production of the key biochemical building block, mevalonic acid. We introduced a simple framework that captures population dynamics of engineered production strains. We demonstrated in several different host strains that evolution substantially affects population structure over industrial timescales, with direct ramifications for bio-based process performance. While ultra-deep-sequencing has advanced the understanding of heterogeneities in human disease evolution 51 , so far no studies have investigated the potential for biotechnological evolution at population depth, in part possibly due to difficulties in resolving structural variations by short-read sequencing of populations. We observed that pathway error modes are dominated by a broad spectrum of IS insertions in non-canonical target sites. These remove or alleviate the production load, and the error modes differ between strains and clone banks, but appear rapidly in a population. We find that two key parameters influence the probability and speed by which evolution can impact cell factory stability: the escape rate, which is the rate by which non-producing mutants are generated in a population; and the production load, which manifests a lower fitness of producing cells in direct competition with non-producers. Based on these parameters, a two-state mathematical model accurately describes the essential population dynamics of experimentally simulated fermentations. By de-convoluting stability into its two principal parameters, the model provides a quantitative framework for evaluating the scale-up process, such as the long-term impact of a loaded pathway enzymatic step. This model describes genetic heterogeneity at the population level and assumes a two-state transition from producer to non-producer cell, which may not be adequate, e.g., for pathways operating with several independently loaded biosynthetic genes. Further, the model assumes growth without nutrient limitations and a constant escape rate and production load. Average experimental estimations appear to approximate the load well in our setup (Supplementary Fig. 1 ), and may help isolate the production escape rate averages. However, escape rates may be stimulated by different molecular stresses, e.g., in the final phase of bioreactor growth, which are unaccounted for in our simulation. In this study, we have approximated the industrial use of gradually increasing seed train sizes under which most cell divisions occur, by strict passing of cultures in exponential phase throughout the study and shown good fit to a simple model. Thus, integration of dynamic models with time-resolved phenotypic and genotypic data may help guide an investigator to separate load and mutational effects during production strain and process development to more rationally accommodate evolutionary process limitations. Our results offer a potential explanation of why lab-scale yields and titers might not accurately predict large-scale fermentation performance following a scale-up procedure, despite vector maintenance. The observed pathway disruptions occur within a plasmid population maintained by selection. We find that these modeled dynamics of structural pathway disruption are similar to those of segregational plasmid loss 33 , although they act at different rate scales and are characterized by a diversity of error modes (Fig. 4 ). As an example, our model predicts that an initially pure producing population, with a production load of 30% and an escape rate of 10 −7 /generation, will shift to 96% non-producing cells over 60 generations, corresponding to a bioreactor of 2 m 3 . Remarkably, the same population at lab-scale (age of 37 generations) might appear high performing with <3% non-producers in the population. Because the majority of product is synthesized when the fermentation population reaches the final density in industrial-scale production, it is crucial to investigate the population genotype at this point and not simply extrapolate phenotypic performance from lab-scale experiments. Time-lapse ultra-deep-sequencing represents a valuable approach for determining error modes, occurrence rates, and their alleviated loads at an early stage. Such ultra-deep-sequencing may also be applied to existing scaled-up fermentations, previously thought to be free of genetic heterogeneity. Common industrial practice employs production strain clone banks stocked as frozen aliquots. Genetic errors in only a few cells might reside in the starting seed although the population appears healthy for a considerable number of divisions. Seeding fermentations from the same cell bank clone therefore generates a highly recurring stability profile. Cell bank aliquots should therefore be evaluated for rare pre-existing mutations that disrupt production and could be selected for during production scale-up. Deep-sequencing of master cell bank aliquots could be applied to test for this. Considering that production load improvements by even a few percent can substantially improve stability (Fig. 2c ), the specific contributors to production loads must be addressed for each pathway and host cell considering a final large-scale process. For example, reducing the production load from 28 to 23% should extend the production half-life by 10 generations (assuming a constant escape rate of 2.1 × 10 –7 /generation). Practical strategies will be required to reduce factors of production load, including medium optimization, improved balancing of pathway gene expression, and the cellular export of toxic by- or end products. Poor balancing may favor accumulation of toxic pathway intermediates, which carry particularly high potential as a production load. Because intermediates are intracellular, associated toxicities selectively target the producing cells. In this case, adding genes to degrade toxic by-products or to dynamically redirect pathway flux and the use of specific metabolite- or stress-induced pathway promoters may be advantageous for limiting production load 52 – 54 . In the rare cases of growth rate-coupled production, semi-continuous processes have been commercialized to improve productivity, such as for R-lactic acid in Lactobacillus 55 . In attempts to improve stability, systems for maintaining metabolic pathways through multiple chromosomal integrations are often used 56 , 57 while stabilizing duplications may also result randomly during optimization 58 . Still, integrated pathways remain subject to intra-pathway disruptions by SNPs, mobile genetic elements, and illegitimate recombination, but independence of the individual integration sites means that individual escapes will not be enriched within the intracellular pathway population such as uneven segregation allows in multi-copy plasmid systems. Independently integrated pathway copies may thus provide stabilization in addition to easier antibiotic-free pathway maintenance, which today appears as the major advantage of chromosomal propagation. Yet multiple targeted integrations require significantly longer construction protocols, especially at >50 copies and when limited to IP-free engineering. Future studies should investigate the changes in intra-pathway escape dynamics of such multi-copy, chromosomally integrated production pathways. Based on our results, removal of mobile elements from the genomes of microbial production strains 59 is a relevant first step for long-term stabilization and the enabling of toxic and burdened pathway expression. Such strategies postpone the onset of significant genetic heterogeneity (Fig. 5c ). Mechanistically, escape via homologous recombination points can be avoided, e.g., by synonymous codons 60 . Alternatively, coupling of essential genes to the production pathway operon can also increase production stability (Fig. 6b ). Dynamic fermentation population models might serve as technical tools to predict the necessary reduction in escape rate or production load for a particular bioreactor size. Knowledge of stability dynamics should ensure a more holistic evaluation of strains by taking into account the potential for rapid performance loss. By characterizing and modeling the interplay between spontaneous genetic errors at depth and their selection by metabolic burden and pathway toxicity, we have shown the paths for their synergistic impact on pathway stability. Furthermore, we have demonstrated how engineered reductions in both production load and escape rate can improve stability. We expect that the results, methodologies, and their implications will open new opportunities for metabolic engineers in the quest to develop sustainable and industrially scalable bioprocesses."
} | 3,988 |
28195581 | PMC5398378 | pmc | 6,189 | {
"abstract": "Thaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 m m of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca . N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1–2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota , a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ . However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota , suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.",
"introduction": "Introduction Nitrification is an important process for municipal and industrial wastewater treatment plants (WWTPs) because it prevents the negative impacts of releasing ammonia to receiving waters, including toxicity to fish, eutrophication and increased oxygen demand. Ammonia-oxidizing bacteria (AOB) were traditionally believed to mediate ammonia oxidation in soils, aquatic habitats and engineered environments. However, several studies have implicated recently discovered ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota as the dominant ammonia oxidizers in many environments, including the open ocean ( Wuchter et al. , 2006 ), soils ( Leininger et al. , 2006 ; Stopnisek et al. , 2010 ; Yao et al. , 2011 ; Zhang et al. , 2012 ) and engineered environments such as aquaculture operations and aquarium biofilters ( Sauder et al. , 2011 ; Brown et al. , 2013 ; Bagchi et al. , 2014 ). However, there is evidence that AOB are numerically dominant in some environments, and that AOB may mediate ammonia oxidation in several soils, despite a numerical dominance of AOA ( Di et al. , 2009 ; Banning et al. , 2015 ; Sterngren et al. , 2015 ). The role of AOA detected in WWTPs remains unclear. Compared with many natural environments, WWTPs contain relatively high levels of ammonia, which should favor AOB over AOA ( Martens-Habbena et al. , 2009 ; Schleper, 2010 ). Indeed, many studies have reported a numerical dominance of AOB in municipal and industrial WWTPs ( Wells et al. , 2009 ; Mussmann et al. , 2011 ; Gao et al. , 2013 ). Nevertheless, AOA have been detected in several WWTPs ( Park et al. , 2006 ; Zhang et al. , 2009 ; Gao et al. , 2013, 2014 ), and in some cases outnumber AOB ( Kayee et al. , 2011 ; Bai et al. , 2012 ). Although the abundance and diversity of AOA and AOB have been assessed in several WWTPs, no previous studies have analyzed the relative contributions of these groups to nitrification in any municipal WWTPs. Intact polar lipids originating from thaumarchaeol have been identified in WWTP biofilms ( Sauder et al. , 2012 ), indicating that the detected Thaumarchaeota were viable in the system, though not necessarily oxidizing ammonia. Mussman et al. (2011) were unable to demonstrate bicarbonate assimilation by Thaumarchaeota in nitrifying sludge and called into question a strictly chemolithoautotrophic lifestyle of thaumarchaeotes in the examined industrial WWTP. Although all cultured members of the Thaumarchaeota oxidize ammonia and fix inorganic carbon autotrophically, their metabolism in the environment may be more complex. AOA genomes encode transporters for a variety of organic compounds (for example, Hallam et al. , 2006 ; Walker et al. , 2010 ; Blainey et al. , 2011 ; Spang et al. , 2012 ) and archaea in the ocean incorporate amino acids ( Ouverney and Fuhrman, 2000 ). In addition, supplementation of AOA cultures with pyruvate and alpha-ketoglutarate stimulates growth ( Tourna et al. , 2011 ; Qin et al. , 2014 ), although this arises in several cultures from non-enzymatic detoxification of hydrogen peroxide ( Kim et al. , 2016 ). Although several Group I.1a Thaumarchaeota representatives have been reported in laboratory cultures (for example, Könneke et al. , 2005 ; Jung et al. , 2011 ; Mosier et al. , 2012 ; Lebedeva et al. , 2013 ; Li et al. , 2016 ), comparatively few Group I.1b AOA have been cultivated. Group I.1b representatives include Nitrososphaera viennensis , Nitrososphaera evergladensis , Nitrosocosmicus franklandus and Nitrosocosmicus oleophilus , which originate from soils or sediments ( Tourna et al. , 2011 ; Zhalnina et al. , 2014 ; Lehtovirta-Morley et al. , 2016 ; Jung et al. , 2016 ), and Nitrososphaera gargensis , which originates from a hotspring ( Hatzenpichler et al. , 2008 ; Palatinszky et al. , 2015 ). A phylogenetic analysis of archaeal amoA gene sequences demonstrated five major AOA lineages, represented by the genera Nitrosopumilus , Nitrosocaldus , Nitrosotalea, Nitrososphaera , and a fifth clade that forms a sister cluster to the Nitrososphaera ( Pester et al. , 2012 ). This clade contains most amoA sequences obtained from WWTPs (for example, Mussmann et al. , 2011 ; Limpiyakorn et al. , 2011 ; Sauder et al. , 2012 ), manure composting facilities ( Yamamoto et al. , 2010 ), landfill sites ( Im et al. , 2011 ) and human skin ( Probst et al. , 2013 ). Only one AOA enrichment culture exists from a wastewater treatment system, belonging to the Group I.1a Thaumarchaeota ( Li et al. , 2016 ). Here, we report the cultivation and characterization of a novel group I.1b Thaumarchoaeta representative, belonging to the Nitrososphaera sister cluster. This representative was enriched from biofilm of rotating biological contactors (RBCs) of a municipal WWTP in Guelph, Canada, where it was first discovered based on DNA and lipid signatures ( Sauder et al. , 2012 ). We propose the name Ca. Nitrosocosmicus exaquare G61 for this representative.",
"discussion": "Discussion Little is known about the metabolism and activity of Thaumarchaeota detected in WWTPs, despite the importance of these environments to human and environmental health. Here, we report the cultivation of a Thaumarchaeota representative originating from a municipal WWTP, which oxidizes ammonia, fixes inorganic carbon, and possesses a genomic repertoire consistent with chemolithoautotrophy. The genus name is based on the related organisms Ca . N. franklandus ( Lehtovirta-Morley et al. , 2016 ) and Ca. N. oleophilus ( Jung et al. , 2016 ), and the species name ‘ exaquare ’ (latin for ‘water running out’ or ‘sewage’) reflects its wastewater origin. Ca . N. exaquare produces nitrite from ammonia at near-stoichiometric values ( Figure 1a ), and thaumarchaeotal cell numbers follow nitrite production closely, providing evidence that energy for cell growth is derived from the oxidation of ammonia to nitrite. The 51.7 h generation time of Ca . N. exaquare is similar to that originally reported for N. viennensis (46 h), although a shorter generation time of 27.5 h was later reported ( Stieglmeier et al. , 2014 ). Ca. N. exaquare is mesophilic, with optimal growth observed at 33 °C ( Figure 1b ). No growth was observed for Ca. N. exaquare above 40 °C, in contrast to N. gargensis , which grows optimally at 46 °C ( Hatzenpichler et al. , 2008 ) and N. viennensis , which can tolerate temperatures of at least 47 °C ( Tourna et al. , 2011 ). Ca. N. exaquare can withstand relatively high concentrations of both ammonia and nitrite ( Figures 1c and d ). Ammonia oxidation proceeded in the presence of up to 15 m m NaNO 2 , with complete inhibition observed at 30 m m . N. viennensis oxidizes ammonia with little inhibition at 10 m m NaNO 2 , but ammonia oxidation ceased if ~3.5 m m nitrification-derived nitrite accumulated ( Tourna et al. , 2011 ). In contrast, Ca. N. exaquare fully oxidizes at least 15 m m NH 4 Cl, indicating that it may be better able to tolerate nitrite or other ammonia oxidation intermediates. Initiation of ammonia oxidation can be achieved with ammonia concentrations of up to 20 m m , with complete inhibition not observed until 30 m m ( Figure 1d ). For the growth conditions used (that is, pH 8, 30 °C), 20 m m NH 4 Cl is equivalent to 1.49 m m un-ionized ammonia (NH 3 ). For comparison, reported inhibitory concentrations of un-ionized ammonia are only 18–27 μm, <9 μ m and 0.51–0.75 μ m for N. maritimus , Ca. N. devanaterra and N. viennensis , respectively (see Hatzenpichler, 2012 for a review). High tolerance to ammonia is perhaps unsurprising given that Ca. N. exaquare originates from a municipal WWTP, where ammonia concentrations would be higher than in most naturally occurring soil or aquatic environments. Niche partitioning occurs between AOA and AOB based on ammonia availability ( Erguder et al. , 2009 ; Jia and Conrad, 2009 ; Martens-Habbena et al. , 2009 ; Schleper, 2010 ; Verhamme et al. , 2011 ; Sauder et al. , 2012 ), but given the high diversity of AOA and their global distribution across diverse environments, it is also possible that niche partitioning based on ammonia concentrations also occurs within the Thaumarchaeota. Similarly, nitrite has been suggested as a major driver of niche partitioning for nitrite-oxidizers from the genus Nitrospira ( Maixner et al. , 2006 ). Ca. N. exaquare is the first reported representative of the Nitrososphaera sister cluster originating from a WWTP. Both amoA ( Figure 2 ) and 16S rRNA ( Supplementary Figure S2 ) gene sequences clustered with other Thaumarchaeota from WWTPs, both industrial and municipal, as well as other waste-related environments such as landfills and landfill-contaminated soils. Most detected thaumarchaeotal sequences from WWTPs affiliate with the I.1b soil group, often in the Nitrososphaera sister cluster ( Mussmann et al. , 2011 ; Sauder et al. , 2012 ; Gao et al. , 2013 ; Limpiyakorn et al. , 2013 ). These engineered environments represent comparatively nutrient-rich habitats, characterized by relatively high levels of organic carbon and ammonia. Combined with the observed high tolerance to ammonia and nitrite, this clustering could reflect an adaptation of Ca. N. exaquare-like Thaumarchaeota to high nutrient environments. With cell sizes up to 2 μm in diameter, Ca. N. exaquare is the largest reported member of the Thaumarchaeota . Most observed cells were ~1.3 μm ( Figure 3a ), which is substantially larger than group I.1a Thaumarchaeota (for example, N. maritimus cells are 0.2 μm × 0.7 μm; Könneke et al. , 2005 ). Group I.1b Thaumarchaeota are larger, including N. gargensis and N. viennensis , which have cell diameters of ~0.9 μm and 0.5–0.8 μm, respectively ( Hatzenpichler et al. , 2008 ; Tourna et al. , 2011 ). Although coccoid morphologies have been reported for all group I.1b Thaumarchaoeta , Ca . N. exaquare cells appear smoothly spherical ( Figure 2a ), whereas N. viennensis cells are irregular, with concave areas that appear collapsed into the cell ( Tourna et al. , 2011 ). The cell size and morphology of Ca. N. exaquare closely resemble thaumarchaeotal cells previously detected by CARD-FISH in wastewater sludge samples from industrial WWTPs ( Mussmann et al. , 2011 ). Ca. N. exaquare incorporated bicarbonate into biomass in association with ammonia oxidation ( Figure 3d ) and encodes the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation pathway ( Table 1 ), which is used by all known Thaumarchaeota ( Berg et al. , 2007 ; Berg, 2011 ). Moreover, it has grown in enrichment culture for several years without any externally supplied organic carbon. These data indicate that Ca. N. exaquare combines ammonia oxidation with autotrophic carbon fixation, as expected for a classical ammonia-oxidizing microorganism. Despite this, Ca. N. exaquare is strongly stimulated by the addition of organic carbon ( Figure 4 ), which may indicate a mixotrophic metabolism, or an indirect benefit. A variety of organic carbon sources accelerated ammonia-oxidizing activity by Ca. N. exaquare, with malate and succinate resulting in the highest level of stimulation ( Figure 4 ). Ca. N. exaquare may be able to incorporate these metabolic intermediates directly into its tricarboxylic acid cycle, which could provide reducing power or precursors for biomolecule synthesis. However, given the wide variety of stimulatory compounds (for example, glycerol, yeast extract, butyrate), it is likely that not all organic carbon sources directly stimulate growth, but instead provide indirect benefits via remaining heterotrophic bacteria. A mixotrophic lifestyle would be consistent with previous environmental observations. For example, marine archaea assimilate amino acids ( Ouverney and Fuhrman, 2000 ), and radiocarbon analyses of the membrane lipids of pelagic marine Thaumarchaeota indicate that communities are composed of combination of autotrophs and heterotrophs, or a single mixotrophic population ( Ingalls et al. , 2006 ). In addition, N. viennensis and marine thaumarchaeotal strains require pyruvate or α-ketoglutaric acid for optimal growth ( Tourna et al. , 2011 ; Qin et al. , 2014 ), although the mechanism of action of these compounds is detoxification of ROS ( Kim et al. , 2016 ). Although ROS detoxification was not demonstrated with succinate, it is possible that this compound stimulates growth of heterotrophs that in turn detoxify ROS and thereby encourage growth of AOA. However, Ca . N. exaquare encodes a variety of genes that may confer protection from ROS: in addition to several genes shared among many Thaumarchaeota (for example, superoxide dismutase, alkyl hydroperoxide reductase), Ca . N. exaquare encodes a peroxidase, which is unique among sequenced thaumarchaeotal genomes, and a manganese-dependent catalase, which is also present in Ca . N. evergladensis ( Table 1 ). The genome of Ca. N. exaquare encodes two gene copies of a sodium-dependent dicarboxylate transporter (SdcS; Table 1 ), which transports succinate, malate and fumarate ( Hall and Pajor, 2005 , 2007 ). SdcS-type dicarboxylate transporters are also encoded in the genomes of Ca . N. evergladensis ( Zhalnina et al. , 2014 ) and Ca. N. uzonensis ( Lebedeva et al. , 2013 ). Expression of this transporter could provide an explanation for the observed stimulation of Ca . N. exaquare by succinate and malate ( Figure 4 ). In addition to being a tricarboxylic acid cycle intermediate, succinate is a central compound in the 3HB/4HP cycle ( Berg, 2011 ) and could feed directly into this carbon fixation pathway. Labeling studies with Metallosphaera medulla , which also uses the 3HP/4HP cycle ( Berg et al. , 2007 ), demonstrated that the majority of anabolic precursors are derived from succinate ( Estelmann et al. , 2011 ). Given the presence of this transporter and the strong stimulatory effects of succinate and malate, C 4 compounds may have an important role in supplementing Ca. N. exaquare metabolism. At 2.99 Mbps, Ca . N. exaquare encodes the largest reported AOA genome, and shares several features with Group I.1b soil Thaumarchaeota ( Table 1 ). Interestingly, Ca. N. exaquare has a G+C content (33.9%) that is lower than other Group I.1b Thaumarchaeota (~50%), but comparable to group I.1a Thaumarchaeota . The genome encodes all key components for ammonia oxidation and bicarbonate fixation pathways, supporting its role as a chemolithoautotrophic ammonia oxidizer. In addition, Ca. N. exaquare has a similar metabolic profile to other AOA ( Figure 5 ), with few genes associated with carbohydrate catabolism. An encoded pathway for degradation of mannosylglycerate was identified as unique among thaumarchaeotal genomes, but most likely relates to osmostic regulation, which has been suggested previously ( Spang et al. , 2012 ; Zhalnina et al. , 2012 ). Ca. N. exaquare also encodes genes associated with C 1 metabolism, including formate dehydrogenase and glutathione-dependent formaldehyde dehydrogenase. Several autotrophic NOB can oxidize or assimilate formate ( Malavolta et al. , 1962 ; Van Gool and Laudelout, 1966 ; Gruber-Dorninger et al. , 2015 ; Koch et al. , 2015 ). However, these encoded enzymes could be used for detoxification, and further work is necessary to assess whether C 1 substrates could supplement autotrophic metabolism. AOA have been detected and quantified in several WWTPs, but only one study has assessed the relative contributions of ammonia-oxidizing prokaryotes to ammonia oxidation in WWTPs. Mussman et al. (2011) detected Thaumarchaeota in industrial WWTPs treating oil refinery waste but found no evidence for bicarbonate fixation, despite active expression of amoA genes. These Thaumarchaeota are phylogenetically related ( Figure 2 ) and morphologically similar to Ca. N. exaquare, which oxidizes ammonia, assimilates bicarbonate and encodes a genome supporting chemolithoautotrophic metabolism. However, ammonia monooxygenase substrate promiscuity has been reported ( Pester et al. , 2011 ), and different growth conditions elicit different physiological responses, so the role of Ca. N. exaquare in situ is likely more complex in natural environments. Incubations of Guelph WWTP RBC biofilm with differential inhibitors indicated that ATU was highly inhibitory, octyne had little effect and PTIO was partially inhibitory. More inhibition by PTIO was observed in RBC 8 biofilms compared with RBC 1 of the same treatment train, suggesting that a larger proportion of the ammonia-oxidizing activity results from AOA. This is supported by qPCR data demonstrating that thaumarchaeotes comprise a higher proportion of the putative ammonia-oxidizing prokaryotes in RBC 8 compared with RBC 1 ( Figure 6; \n Supplementary Table S5 ). Octyne and ATU are specific inhibitors of AOB ( Hatzenpichler et al. , 2008 ; Shen et al. , 2013 ; Taylor et al. , 2013 ) and results obtained from these compounds should ideally be similar, but were inconsistent in this study. Several advantages have been reported for octyne ( Taylor et al. , 2013 ), but it has not been used previously with samples from a WWTP environment, and may have been degraded by biofilm microorganisms. Two PTIO concentrations were included because lower concentrations may be insufficient in environmental samples due to production of nitric oxide from non-nitrification processes (for example, denitrification), but higher concentrations might result in inhibition of some AOB. For example, PTIO concentrations of 400 μ m are partially inhibitory to N. multiformis ( Shen et al. , 2013 ), although not to N. europaea ( Supplementary Figure S9 ). Although questions remain regarding the efficacy of octyne at inhibiting AOB in this biofilm, and whether AOB were also inhibited using 400 μ m PTIO, the observed inhibition of ammonia-oxidizing activity by 200 μ m PTIO suggests that Thaumarchaeota contribute to ammonia-oxidizing activity of the biofilm. The previously reported relationship between ammonia concentration and thaumarchaeotal abundance in this biofilm ( Sauder et al. , 2012 ) supports the role of Ca . N. exaquare-like AOA as ammonia oxidizers in situ . Although this work only considers AOA and AOB, completely nitrifying Nitrospira organisms (that is, comammox bacteria; van Kessel et al. , 2015 ; Daims et al. , 2015 ), with unknown sensitivities to these inhibitors, could be contributing to nitrification activity in the biofilm. The CARD-MAR-FISH data from the RBC biofilm samples indicated that when supplied with ammonia, both AOB and Nitrospira are MAR-positive, whereas there was no evidence for bicarbonate fixation by Thaumarchaeota ( Figure 7 ). This suggests that the detected Thaumarchaeota cells were either predominantly relying on an alternative metabolism, or that they were oxidizing ammonia for energy but assimilating a carbon source other than bicarbonate. Similarly, related Thaumarchaeota in another WWTP did not assimilate inorganic carbon in the presence of ammonia ( Mussmann et al. , 2011 ). Most observed AOB showed strong MAR signals, which is consistent with the strongly inhibitory effect of ATU. Positive MAR signals were observed for some Nitrospira microcolonies ( Figure 7 ), which could have arisen from either nitrite-oxidizing or comammox activity. Ca. N. exaquare is the first group I.1b Thaumarchaeota representative cultivated from a WWTP, and clusters phylogenetically with AOA originating from wastewater environments. The laboratory activity and genetic complement of Ca. N. exaquare suggest that it is a classic ammonia-oxidizing microorganism, which may be stimulated by organic carbon. In the wastewater biofilm from which it originates, both qPCR and qFISH indicate that Thaumarchaeota consistently outnumber AOB. However, the metabolic role played in situ by Ca . N. exaquare appears to be more complex than strictly chemolithoautotrophic nitrification, and it is possible that the relatively high abundance of Thaumarchaeota could be explained by growth on other substrates present in the biofilm. Further work is needed to elucidate the contributions of Ca . N. exaquare-like Thaumarchaeota to ammonia oxidation in this system and to assess the in situ potential for autotrophic or mixotrophic metabolisms."
} | 5,653 |
23216788 | null | s2 | 6,190 | {
"abstract": "The relationship between ecological variation and microbial genetic composition is critical to understanding microbial influence on community and ecosystem function. In glasshouse trials using nine native legume species and 40 rhizobial strains, we find that bacterial rRNA phylotype accounts for 68% of amoung isolate variability in symbiotic effectiveness and 79% of host specificity in growth response. We also find that rhizobial phylotype diversity and composition of soils collected from a geographical breadth of sites explains the growth responses of two acacia species. Positive soil microbial feedback between the two acacia hosts was largely driven by changes in diversity of rhizobia. Greater rhizobial diversity accumulated in association with the less responsive host species, Acacia salicina, and negatively affected the growth of the more responsive Acacia stenophylla. Together, this work demonstrates correspondence of phylotype with microbial function, and demonstrates that the dynamics of rhizobia on host species can feed back on plant population performance."
} | 270 |
32218779 | PMC7079680 | pmc | 6,194 | {
"abstract": "Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of synthesis gas (syngas), for autotrophic growth in order to produce biomass and metabolites such as acetyl-CoA via the Wood–Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution (ALE) for growth improvement of Eubacterium limosum ATCC 8486 under high CO conditions. The strain evolved under syngas conditions with 44% CO over 150 generations, resulting in a significant increased optical density (600 nm) and growth rate by 2.14 and 1.44 folds, respectively. In addition, the evolved populations were capable of proliferating under CO concentrations as high as 80%. These results suggest that cell growth is enhanced as beneficial mutations are selected and accumulated, and the metabolism is altered to facilitate the enhanced phenotype. To identify the causal mutations related to growth improvement under high CO concentrations, we performed whole genome resequencing of each population at 50-generation intervals. Interestingly, we found key mutations in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding genes, acsA and cooC . To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth rate and CO tolerance level of the single clone were comparable to those of the evolved populations and wild type strain under CO conditions. Furthermore, the evolved strain produced 1.34 folds target metabolite acetoin when compared to the parental strain while introducing the biosynthetic pathway coding genes to the strains. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486.",
"introduction": "Introduction Carbon monoxide (CO), generated due to incomplete combustion of organic materials, is a toxic gas that hampers the growth of various organisms. Presently, CO is emitted in large quantities in the form of synthesis gas (syngas) comprising CO, carbon dioxide (CO 2 ), and hydrogen (H 2 ). The syngas is produced as a byproduct of fossil fuel combustion for industrial development, specifically by gasification of coal, biomass, and natural gas. The syngas composition depends on the gasifier type and resource, which increases the CO amount up to 67% of the total volume ( Subramani and Gangwal, 2008 ; Munasinghe and Khanal, 2010 ). Being derived from fossil fuel, syngas needs to be purified in order to prevent air pollution and the greenhouse gas effect, which is conventionally managed via thermochemical processes that convert syngas into liquid hydrocarbons. Unfortunately, the conventional method requires greater operation cost and high temperature and pressure conditions, thus requiring a more efficient method to convert syngas into other chemicals ( Bredwell et al., 1999 ; Munasinghe and Khanal, 2010 ). As an alternative method, gas fermentation using microorganisms has been suggested to produce industrial commodities with lower operation cost and higher catalyst specificity compared to the thermochemical processes. In addition, the biological process is capable of producing various organic compounds using syngas as feedstock, such as acetate, butyrate, ethanol, butanol, 2,3-butanediol, and other compounds via genetic manipulation ( Köpke et al., 2010 , 2011 ; Abubackar et al., 2015 ; Park et al., 2017 ). Among the promising biocatalysts for syngas fermentation, with an ability to convert CO into biomass and various biochemicals, acetogenic bacteria (acetogens) have received immense attention and are considered as a novel platform to replace the conventional processes ( Henstra et al., 2007 ; Bengelsdorf et al., 2013 ; Latif et al., 2014 ). Acetogens are anaerobic bacteria that utilize CO and CO 2 as a carbon building block and, initially, synthesize acetyl-CoA as an important metabolic intermediate, by using the Wood–Ljungdahl pathway (WLP) ( Drake et al., 2008 ). The linear WLP comprises two branches, methyl and carbonyl branches, which convert CO into CO 2 and then to acetyl-CoA ( Drake et al., 2006 ). The methyl branch reduces CO 2 converted from CO into formate, catalyzed by formate dehydrogenase ( fdh ) ( Ragsdale, 1997 ). Following the initial reaction, formyl-tetrahydrofolate (THF) is formed using formate and THF, which requires the hydrolysis of ATP ( Schuchmann and Müller, 2014 ). Subsequently, the formyl-THF is converted by methenyl-THF cyclohydrolase into methenyl-THF, and further into methylene-THF via methylene-THF dehydrogenase. Eventually, the methyl branch reduces methylene-THF to methyl-THF by methylene-THF reductase ( Ragsdale and Pierce, 2008 ). For the carbonyl branch, the methyl group of methyl-THF is transferred by methyltransferase to corrinoid Fe-S protein, and then to CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), which carries CO generated by using CODH/ACS from CO 2 ( Ljungdahl, 1986 ). The condensation of methyl group and CO from the methyl and carbonyl branches, respectively, generates acetyl-CoA, which then converts into acetate by generating ATP ( Ragsdale and Pierce, 2008 ). Of all the enzymes associated with the WLP, CODH/ACS plays a pivotal role in autotrophic growth of acetogens by reversibly interconverting CO/CO 2 and synthesizing acetyl-CoA ( Doukov et al., 2002 ). In acetogens, the CODH/ACS complex is formed by the assembly of ACS and CODH, as (αβ) 2 complex in the presence of [3Fe-4S] cluster (C-cluster), [4Fe-4S] cluster (A-cluster), and metal clusters as the active sites ( Darnault et al., 2003 ; Ragsdale, 2008 ; Appel et al., 2013 ; Can et al., 2014 ). The C-cluster encoded by acsA is the active site of CODH subunit for reversible oxidation of CO to CO 2 ( Doukov et al., 2002 ). The A-cluster encoded acsB is the active site of ACS subunit, which generates acetyl-CoA from CO, CoA, and methyl group that is transferred from the corrinoid protein ( Seravalli et al., 1997 ; Darnault et al., 2003 ; Drennan et al., 2004 ). For CO fixation of Moorella thermoacetica , for example, CO catalytic reaction is indicated as “ping-pong” reaction involving two steps, ping and pong step ( Diekert and Thauer, 1978 ). In the ping step, CO binds to the metal center of the C-cluster, which is nickel, and thus reduces the C-cluster; thereafter, in the pong step, the electrons from the C-cluster are transferred to the external electron acceptors, such as ferredoxin, via the B- and D-clusters, and CO 2 is generated by CO oxidation ( Can et al., 2014 ). To activate this complex, specific accessory proteins, such as cooC , cooJ , or cooT , are required, which are responsible for binding the metal and forming metal binding site for the complex interface ( Bender et al., 2011 ; Alfano et al., 2019 ). In addition, the proteins support maturation of CODH by assembling C-cluster in the CODH/ACS complex and transfer electrons obtained from CO oxidation to the electron carriers ( Loke and Lindahl, 2003 ). Using the WLP and the associated enzymes, acetogens utilize CO as a carbon substrate for producing biomass building blocks; however, they are inhibited by the high concentration of CO ( Daniel et al., 1990 ). CO competitively binds to the active site of metalloenzyme, such as hydrogenase, and depletes the transition metal that leads to insufficient ligation of the original substrate, and the absence of metals causes low growth rate and eventually leads to mortality of the organism ( Bertsch and Müller, 2015 ). For example, in Acetobacterium woodii , one of the well-known model acetogens, the growth rates under autotrophic growth conditions decreased with increasing CO concentrations, which also affected the heterotrophic growth conditions ( Bertsch and Müller, 2015 ). CO inhibited hydrogen-dependent CO 2 reductase of A. woodii , which is responsible for CO 2 reduction and hydrogen storage ( Bertsch and Müller, 2015 ). Although acetogens utilize CO as the carbon source, the inhibitory effect of high CO concentration on the growth and lethality of the organisms need to be enhanced for efficient CO fixation. In the present study, we applied adaptive laboratory evolution (ALE) method to enhance CO tolerance and growth fitness of Eubacterium limosum ATCC 8486 under CO presence, by serially transferring the strain on syngas containing 44% CO for 150 generations. ALE is widely utilized, thus allowing self-optimization of the organism to acquire the desired phenotype ( Elena and Lenski, 2003 ; Dragosits and Mattanovich, 2013 ; Choe et al., 2019 ). Genome sequencing of the evolved strains at 50-generation intervals revealed several causal mutations, which were identified in the genes encoding CODH/ACS. Subsequently, via the growth profiling of single isolated clone under syngas growth conditions, we validated that the key mutation altered the tolerance and the growth of the strain. The results provide insights on CO fixation for strain designing.",
"discussion": "Discussion Converting CO into biofuels and biochemicals provides several advantages such as low feedstock cost, utilization of harmful gas, and reduction of climate-changing substrate. Despite the advantages, CO has been a challenging feedstock due to lack of suitable microorganisms that tolerate and utilize CO as a substrate. Among the candidate organisms, acetogens have been suggested as a crucial platform to convert CO into various biochemicals using the WLP that oxidizes CO into CO 2 , and then into an important major metabolite, acetyl-CoA. All acetogens carry the unique pathway to synthesize acetyl-CoA under autotrophic growth conditions; however, the growth is inhibited under high CO concentrations, indicating that CO utilizing acetogens are intolerant toward CO. According to the previous studies, growth of A. woodii , a model acetogen that is phylogenetically related to E. limosum , is completely inhibited by the presence of CO with higher than 25% in the culture headspace ( Bertsch and Müller, 2015 ). In this study, using ALE, the growth rate and CO tolerance of E. limosum were enhanced, which is highly related with the previous studies on Thermoanaerobacter kivui and Butyribacterium methylotrophicum . The studies reported that passaging the strains under CO conditions few times enhanced the utilization of CO as a feedstock, suggesting that wild type acetogens under CO conditions are not optimal and further enhancement is possible via ALE ( Lynd et al., 1982 ; Weghoff and Müller, 2016 ). The enhanced growth rate of E. limosum was higher than that of other acetogens under CO conditions, including the growth rates of the adjusted acetogens ( Table 2 ). Prior to this study, the highest growth rate under CO growth condition was that of Clostridium carboxidivorans (0.084 h –1 ), followed by T. kivui (0.068 h –1 ) and C. ljungdahlii (0.060 h –1 ), which are known as CO utilizing organisms ( Phillips et al., 1994 ; Fernandez-Naveira et al., 2016 ; Weghoff and Müller, 2016 ). The growth rates of E. limosum under CO condition changed from 0.058 to 0.089 h –1 and increased by 1.53 folds through the ALE, and the growth rate under 100% CO condition was 0.048 h –1 , thus indicating that E. limosum is one of the fastest growing acetogen strains under the autotrophic growth condition. TABLE 2 Comparison of the growth rates of acetogens. Strain Gas condition Growth rate References Acetobacterium woodii 5% CO/16% CO 2 /64% H 2 (100 kPa) 0.028 h –1 Bertsch and Müller, 2015 10% CO/16% CO 2 /64% H 2 (100 kPa) ∼ 0.022 h –1 15% CO/16% CO 2 /64% H 2 (100 kPa) ∼ 0.011 h –1 25% CO/15% CO 2 (100 kPa) No growth Butyribacterium methylotrophicum 100% CO (100 kPa) 0.050 h –1 Lynd et al., 1982 Clostridium autoethanogenum 45% CO/20% CO 2 /2% H 2 (200 kPa)* 0.057 ± 0.04 h –1 Marcellin et al., 2016 100% CO (200 kPa) 0.019 h –1 Liew et al., 2016 Clostridium carboxidivorans 100% CO (120 kPa) 0.084 ± 0.004 h –1 Fernandez-Naveira et al., 2016 Clostridium ljungdahlii 80% CO/20% CO 2 (200 kPa) 0.060 h –1 Phillips et al., 1994 Eubacterium limosum 44% CO/22% CO 2 /2% H 2 (200 kPa)* 0.095 ± 0.000 h –1 This study Moorella thermoacetica 30% CO/30% CO 2 (240 kPa) 0.069 h –1 Daniel et al., 1990 Thermoanaerobacter kivui CO 20% (200 kPa) Makeup gas (80% N 2 /20% CO 2 ) 0.037 h –1 Weghoff and Müller, 2016 CO 50% (200 kPa) Makeup gas (80% N 2 /20% CO 2 ) 0.045 h –1 CO 70% (200 kPa) Makeup gas (80% N 2 /20% CO 2 ) 0.068 h –1 CO 90% (200 kPa) Makeup gas (80% N 2 /20% CO 2 ) 0.020 h –1 CO 100% (200 kPa) 0.021 h –1 *Industrial syngas composition. Understanding the genomes of phenotypically altered organisms reveals a relationship between the genotype and phenotype. Genome resequencing of the evolved E. limosum identified five key mutations sites in the genome. Specifically, mutations on acsA and cooC2 were in accordance with the previous understanding on CO oxidization mechanism that CODH/ACS complex plays a vital role under the CO fixing condition. However, the mutation on cooC2 , which is crucial for CO oxidation by activating CODH/ACS protein complex by binding to the essential metals, contradicts our hypothesis by introducing an early stop codon at 20 sequences downstream of the mutation site, thus reducing the protein comprising 261 amino acids to 92 amino acids. The insertion of early stop codon prevents translation of the cooC2 active site that is located at Cys116 and Cys118, which are the conserved sites for metal binding, leading to loss of function. In the previous study ( Merrouch et al., 2018 ), increase in the cooC expression elevated CODH activity only in media without nickel supplementation; however, in the present study, nickel was supplemented in the media, making cooC2 unessential under the condition that led to an introduction of a stop codon for the loss of function; whereas, a mutation on acsA , which is responsible for C-cluster of CODH/ACS complex, potentially altered the protein structure. In the evolved strain, ECO, metabolite production pattern changed compared to the wild type E. limosum . In the wild type, acetate was majorly produced, which was similar for the ECO. Interestingly, the ECO, with higher growth rate compared to wild type, produced butyrate under CO condition, with acetate as the major metabolite. Despite the similarity at the genomic level between the E. limosum strains (ATCC 8486 and KIST612), butyrate production was not observed for wild type E. limosum ATCC 8486 under the CO condition, thus contradicting the previous report on E. limosum KIST612 ( Chang et al., 1998 ). E. limosum KIST612 produced acetate and butyrate under CO condition ( Jeong et al., 2015 ). For butyrate production, three additional reduction powers are required that recycle the excessive reducing equivalents, with potential ATP production ( Kerby et al., 1997 ). CO oxidation generates reducing equivalent that needs to be oxidized, often utilized for reducing WLP enzymes to convert carbons and pumping ions across the membrane to create a chemiosmotic gradient for ATP synthesis. Based on the phenotypic results, the ECO altered the metabolite pathway to produce butyrate for oxidizing excessive reduction power and generating ATP, which potentially oxidizes CO faster with more available oxidized electron carriers that needs to be further validated ( Supplementary Figure S4 ). Overall, we developed E. limosum strain that tolerates and efficiently utilizes CO as feedstock via ALE, then identified a key mutation on acsA encoding a subunit of CODH/ACS complex that caused phenotypic traits, and thereafter validated the hypothesis through phenotypic assays. Eventually, we utilized the ECO_acsA strain to construct an engineered strain to produce biochemical using CO as carbon source. The results will serve as an important resource for optimizing CO fermentation and strain designing for better biochemical production."
} | 4,045 |
40042259 | PMC11881642 | pmc | 6,195 | {
"abstract": "Plant functional connectivity—the dispersal of plant propagules between habitat patches—is often ensured through animal movement. Yet, there is no quantitative framework to analyse how plant–animal interactions and the movement of seed dispersers influence community-level plant functional connectivity. We propose a trait-based framework to quantify plant connectivity with a model integrating plant–frugivore networks, animal-mediated seed-dispersal distances and the selection of target patches by seed dispersers. Using this framework, we estimated how network specialization, between-patch distance and resource diversity in a target patch affect the number and diversity of seeds dispersed to that patch. Specialized networks with a high degree of niche partitioning in plant–frugivore interactions reduced functional connectivity by limiting the diversity of seeds dispersed over long distances. Resource diversity in the target patch increased both seed number and diversity, especially in specialized networks and within short and intermediate distances between patches. Notably, resource diversity was particularly important at intermediate distances, where the number and diversity of seeds reaching a patch increased more strongly with resource diversity than at longer distances. Using a trait-based framework, we show that resource diversity in the target patch is a major driver of connectivity in animal-dispersed plant communities.",
"introduction": "1 . Introduction Plant functional connectivity refers to the dispersal of plants and their genes between habitat patches [ 1 , 2 ]. High connectivity promotes the recolonization of habitat patches where plants have gone locally extinct and is essential to the restoration of plant communities [ 3 , 4 ]. For most plants, the movement underpinning this connectivity happens through seed dispersal by animals [ 5 ]. In the tropics, 75–90% of plant species depend on animals for seed dispersal [ 2 , 6 ]. Plant and animal traits shape the occurrence and frequency of frugivory and seed-dispersal interactions [ 7 , 8 ] and can be used to estimate distances of animal-mediated seed dispersal for entire plant communities (e.g. [ 9 , 10 ]). Despite the importance of animals for seed dispersal, there is still no comprehensive framework that allows a mechanistic understanding of how the combined effects of plant–animal interactions and animal movement shape animal-mediated plant functional connectivity [ 11 , 12 ]. Such a framework can be a valuable tool to reveal how plant–animal interactions and animal movement together drive connectivity and influence the restoration of plant communities. Plant dispersal is intertwined with the movement ecology of their dispersal vectors [ 5 ] and can be separated into two components: the vector definition and the actual movement. In animal-mediated seed dispersal, the vector can be determined by an animal selecting certain fruits [ 13 , 14 ]. Plant dispersal, i.e. seed dispersal from point A to point B, depends on the distance and direction of the seed dispersing animal’s movement. Both movement distance and direction are often constrained by the animal’s motion capacity, which is usually related to its morphological traits [ 15 , 16 ]. Both also relate to navigation and the animals’ decision of where to target its movement [ 17 , 18 ]. This decision is influenced by the animal’s internal state, which drives movement based on specific goals, such as foraging movements to preferred resources [ 17 , 19 ]. Integrating movement and functional ecology into seed-dispersal studies (e.g. to estimate seed-dispersal distances as in [ 9 , 10 ]) can offer a deeper understanding of how plant–animal interactions and animal movements influence plant functional connectivity [ 20 , 21 ]. The vectors of seed dispersal can be identified through community-wide interaction networks that can be used to quantify how frequently animal species disperse the seeds of particular plant species. These networks represent species interactions, linking plants to their animal seed dispersers at the level of entire ecological communities. Within these networks, interaction probabilities between plants and animals depend on the compatibility of their traits, especially in the tropics [ 8 , 22 ]. Interaction probabilities are higher when plant and animal species traits closely match [ 14 ]. The degree of trait matching, e.g. the specificity of matching between fruit size and avian gape size, relates to the level of network specialization [ 23 , 24 ]. A high degree of trait matching reduces the number of potential interaction partners, leading to greater niche partitioning among seed dispersers and more exclusive interactions in specialized networks. In contrast, a low degree of trait matching results in more generalized networks, where most plant species share their seed dispersers [ 15 , 25 ]. Based on these trait-matching rules, models can be used to simulate seed-dispersal networks with different degrees of specialization. Seed-dispersal distances may vary across different animal seed dispersers [ 26 ]. Dispersers’ traits that relate to movement ability and seed digestion may affect seed dispersal distances [ 11 , 27 ]. For instance, larger birds tend to retain seeds for a longer period of time and fly faster than smaller birds [ 28 , 29 ], dispersing seeds over longer distances [ 30 ]. By studying seed dispersal by different animal species, models can predict the cumulative distances of all potential seed dispersal events of a plant species that together form a seed-dispersal kernel [ 31 – 33 ]. In combination with interaction networks, trait-based movement models allow the estimation of total seed-dispersal kernels for entire plant communities [ 15 ]. Typically, these kernels show a higher density of dispersal events at shorter distances, with the probability of dispersal decreasing as distance increases [ 33 , 34 ]. Consequently, plant functional connectivity between habitat patches likely decreases with distance, as seeds have lower chances of reaching patches at longer distances. Previous simulations have shown that specialized networks result in shorter community-wide seed dispersal distances than generalized networks, suggesting that large birds that are essential for seed dispersal over long distances contribute less to seed dispersal of the entire plant community in specialized compared to generalized networks [ 15 ]. However, such effects of network specialization on seed-dispersal distances have not been tested in simulations of plant functional connectivity, which additionally depends on how seed dispersers direct their movement to potential target patches. Movement direction is crucial for plant functional connectivity because animals can provide directed dispersal to specific locations [ 35 , 36 ]. An important, but rather little-studied determinant of movement direction is the resource diversity in a habitat patch, which is likely to influence the chances of seed dispersal to patches [ 37 , 38 ] because foraging is one of the primary motivations for frugivore movements [ 27 ]. Frugivores can track resources to minimize foraging time and maximize energy intake [ 39 , 40 ]. At a landscape scale, frugivorous birds are attracted to areas with greater fruit abundance and diversity, and, upon arrival, they tend to forage selectively on specific fruits [ 41 , 42 ]. Therefore, the movement of seed dispersers towards a target patch can be influenced by the abundance of their preferred fruit resources [ 43 ]. For example, larger birds that prefer larger fruits are more likely to occur in habitats with large fruits, while smaller birds are likely to favour patches with smaller fruits [ 44 ]. Thus, the resource diversity in a patch can be used to estimate the probability that frugivores will disperse seeds to that patch. Therefore, patches with a greater diversity of fruiting plants are likely to attract a broader range of animals [ 45 ], which is likely to promote community-level plant functional connectivity. Here, we use a trait-based simulation model to investigate how the movement of avian seed dispersers influences plant functional connectivity across entire plant communities. We built upon established trait-based models and added two important landscape-level aspects (i.e. between-patch distance and resource diversity in the target patch) to these models to test how seed-disperser movement may affect functional connectivity at the plant community level. We focused on birds owing to their high mobility [ 46 ] and their crucial role as seed dispersers [ 47 , 48 ] and leveraged established trait-based models parameterized with bird data. With our model, we tested the previously unexplored interacting effects of network specialization, between-patch distance and resource diversity in the target patch on plant functional connectivity ( figure 1 ). In the simulations, we quantified community-level plant functional connectivity by calculating both the number and the diversity of seeds reaching a target patch from a source patch. We hypothesized that plant functional connectivity decreases with increasing network specialization [ 15 ] and between-patch distance [ 49 ], and that it increases with resource diversity in a target patch. Importantly, we predicted that these three factors are not independent from each other, but interact in their effects on plant functional connectivity. Figure 1 . Quantitative framework for community-level plant functional connectivity. (a) The degree of trait matching between plants and birds defines network specialization in the source patch and the specific dispersal vectors of each plant species [ 25 ]. Interaction probabilities are represented by circle sizes in the resulting matrix; the degree of trait matching is varied by the parameter ‘ s ’ in the trait-matching function [ 24 ]. (b) Seed dispersal distances define reachable habitat patches. Total dispersal kernels for entire plant communities are simulated using avian traits [ 15 ]. In the resulting matrix, black squares indicate potentially successful dispersal events to a target patch. (c) Resource diversity in the target patch defines movement direction. Movement probability towards a target patch is quantified via its relative attractiveness. We hypothesized differences in the relative attractiveness of a target patch for low and high degrees of trait matching. At low trait matching, birds are attracted to many plant species, resulting in a gradual increase in relative attractiveness as resource diversity increases. At high trait matching, birds are attracted by only a few plant species, so relative attractiveness quickly increases once these species are present. In the resulting matrix, grey intensity corresponds to the relative attractiveness of a target patch for a specific bird species. Quantitative framework for community-level plant functional connectivity.",
"discussion": "4 . Discussion We developed a quantitative framework to analyse the interacting effects of network specialization, between-patch distance and resource diversity in the target patch on community-level plant functional connectivity. We found that resource diversity in the target patch generally increased both the number and diversity of dispersed seeds, but that this effect changed with the level of network specialization and distance between patches. Particularly, the effect of resource diversity on connectivity was most pronounced at high levels of specialization and at intermediate distances between patches. Overall, our simulations suggest that diverse plant communities can attract a wider range of seed-dispersing bird species, which is crucial for maintaining high functional connectivity and for promoting forest restoration. In our simulations, we found that lower network specialization increased the diversity of seeds reaching the target patch. While the number of seeds remained almost constant across different levels of specialization, birds in less specialized networks dispersed a more diverse set of plant species to the target patch compared with those in specialized networks (see also [ 15 ]). For generalized networks, we found that even when the resource diversity in the target patch was low or the distance between patches was large, a high diversity of plant species was still dispersed. This raises the chances of successful recruitment and a rapid increase in plant diversity in these patches. In contrast, in specialized networks, this process might be slower owing to a more limited diversity of seeds dispersed to the target patch. Interestingly, the decrease in connectivity with increasing specialization varied with distance. At shorter distances between patches (10 and 50 m), connectivity gradually decreased with specialization level, whereas at larger distances (>100 m), this effect became more pronounced and we observed a sharp decline in connectivity at specialization values larger than 0.3. In real-world networks, specialization levels typically range between 0.2 and 0.5 [ 52 ], suggesting that the low levels of connectivity at distances larger than 100 m may be representative of natural conditions. Our findings also align with previous empirical works that show that generalist bird species play a key role in enhancing plant diversity and habitat connectivity, which benefits plant regeneration in early successional forests [ 48 , 57 ]. While existing studies mostly focus on the dichotomy between forest and matrix habitats [ 12 , 58 ], our simulation quantifies how the specificity of plant–bird interactions may influence the diversity of seeds that can be dispersed between habitat patches. This extends previous empirical work, which has shown that specialized networks can provide higher seed density [ 58 ]. Our framework could also be valuable to explore metacommunity dynamics [ 59 ], offering a tool to test how interaction specificity (either through preference or specialization [ 60 ]), shapes functional connectivity across fragmented landscapes. Furthermore, based on empirical studies of network specialization along large- and small-scale environmental gradients, it may be possible to infer through our simulations that plant functional connectivity may be greater in temperate rather than tropical climates and in the Afrotropics rather than the Neotropics [ 61 , 62 ]. Importantly, generalized plant–frugivore networks at forest edges may help to promote functional connectivity at the landscape scale [ 63 ]. The distance between habitat patches primarily affected the number of seeds dispersed to a target patch and affected seed diversity mostly indirectly via a reduction in seed numbers. The number of seeds arriving at a target patch strongly depended on the resource diversity in the target patch. According to our simulations, the number of seeds dropped significantly even at a between-patch distance of just 10 m if the resource diversity in the target patch was lower than 15 plant species. This highlights the importance of resource diversity for promoting seed dispersal between habitat patches. Because we found similar patterns at 0 and 10 m distances of seed dispersal (electronic supplementary material, figures S3 and S4), similar effects may be expected for seed dispersal within habitat patches. This suggests that seed dispersal within patches is most likely in patches with a high resource diversity. This is important because seed dispersal within patches contributes to plant regeneration and population persistence [ 64 ]. The findings of our simulations are consistent with numerous empirical [ 30 , 49 , 65 ] and simulation studies [ 10 , 54 , 66 ] of avian seed dispersal. For instance, Camargo et al . [ 49 ] reported a significant reduction in seed density from patches located at 10–50 m distance to forest remnants and recorded almost no seeds reaching plots at 300 m distance from the forest source. This is in line with our findings; however, rare long-distance seed dispersal events can be important for plant dispersal to more distant patches and to initiate the recolonization of remote sites [ 34 , 67 ]. Although these events can be crucial for plant populations, they may have little effect on the overall number and diversity of seeds dispersed at the community level. Previous work suggested that short-distance seed dispersal is most relevant for community-level plant functional connectivity and that habitat patches may be crucial as stepping stones in real-world landscapes [ 64 , 68 ]. Our simulations suggest that a high patch density may be required to enhance the functional connectivity of bird-dispersed plant communities because most dispersal events are likely to happen between neighbouring patches. Nevertheless, we caution against an overinterpretation of the specific seed-dispersal distances predicted by our trait-based model because the absolute distances may vary depending on the assumptions of the model and the trait distributions of plant and bird communities. Applying the framework to real-world data is therefore required to validate the prediction of seed-dispersal distances and identify potential distance thresholds for plant functional connectivity. Such empirical studies could also serve to test the relationship between resource diversity and the diversity of seeds arriving at habitat patches of different size (see [ 69 ] for a simulation of seed dispersal in a real landscape). Resource diversity affected both the number and diversity of seeds reaching target patches in our simulations. In particular, in specialized networks and at intermediate distances between patches (50–100 m), a large resource diversity was required to sustain plant functional connectivity because the birds that disperse seeds over these distances are more selective towards their resources in specialized networks. These results align with previous studies showing that resource diversity becomes increasingly important with high levels of specialization [ 45 , 70 ]. At long distances (250 m or more), resource diversity of the target patch had little effect because the number and diversity of seeds that were predicted to be dispersed to long distances were so low that it was largely independent of resource diversity. This suggests that resource diversity plays a prominent role in maintaining plant functional connectivity in landscapes with a high patch density, whereas in more fragmented landscapes, connectivity primarily relies on rare long-distance dispersal events performed by large birds. It is therefore likely that large-fruited plants dispersed by large birds may have better gene flow in highly fragmented landscapes [ 71 ]. Our proposed framework resonates with recent seed-dispersal research that aimed to integrate concepts of fruit tracking and animal movement [ 27 , 72 ] and emphasized the importance of seed-disperser behaviour and seed-dispersal movements for plant community dynamics [ 5 , 21 , 69 ]. While many studies highlight the role of fruit abundance as a key driver of frugivore movements [ 37 , 38 , 47 , 73 ], avian frugivores’ preferential selection of specific fruits [ 60 ] is also a key driver of their movement [ 41 , 74 ]. Our additional analysis, which excludes trait-matching from the calculation of relative attractiveness, shows that resource abundance seems to be the main driver of the number of dispersed seeds [ 75 ]. However, our simulations also demonstrate that plant–animal trait-matching is particularly important in determining the diversity of seeds dispersed into habitat patches. More specifically, we show that plant functional connectivity may critically depend on the diversity of available fruit resources and how these fruits are selected by the seed dispersers. High resource diversity in a target patch may therefore also promote the potential for directed dispersal into such patches [ 76 , 77 ]. By showing the importance of resource diversity, our simulations have important implications for ecological restoration. We found that the functional connectivity of animal-dispersed plant communities critically depends on the diversity of both bird and plant species, and that it can be increased by attracting a diverse set of seed-dispersing bird species to regenerating habitat patches. Active restoration measures, such as planting fruiting trees and shrubs, may therefore significantly assist the regeneration process and help direct animal seed dispersers to regenerating forest ‘islands’ [ 77 – 79 ], especially if plant diversity in such islands is high [ 80 ]. In line with our findings, the inclusion of keystone resources—plants with fruits that are highly attractive to different groups of seed dispersers—is likely to increase the dispersal of seeds into regenerating habitat patches [ 81 , 82 ]. Additionally, planting ‘little islands’ of fruit resources could improve connectivity in highly fragmented landscapes, accelerating forest restoration [ 69 , 83 ]. While our framework focuses on the morphological traits of the plant community, it is important to acknowledge that other traits, such as the nutritional content of the fruits or phenological variation among plant species, significantly affect foraging decisions by frugivores [ 42 , 74 , 84 ] and may influence the plant species reaching target patches [ 49 ]. Future studies on plant functional connectivity should also integrate other types of fruit traits to provide a more comprehensive understanding of the mechanisms driving plant functional connectivity. Moreover, other factors such as forest cover and patch size are known to play important roles in restoration dynamics [ 69 , 85 ]. While these factors may be crucial to explain the context-dependence of restoration dynamics [ 86 , 87 ], our simulations suggest that a high fruit diversity, which is likely to be positively related to patch size [ 88 ], may generally enhance plant functional connectivity. Another important extension in future simulation model studies should be the inclusion of other animal seed dispersers such as frugivorous bats, as these species are often particularly important at early stages of forest recovery [ 89 , 90 ]. By proposing a trait-based framework to assess the functional connectivity of bird-dispersed plant communities, we integrate connectivity concepts from plant and movement ecology. We foresee that the application of our quantitative framework, which comprises all major processes of seed dispersal by animals, can contribute to a more mechanistic understanding of the processes that limit plant functional connectivity in real-world landscapes. The increasing availability of plant and bird trait data [ 91 , 92 ] will make it rather easy to apply the framework to different types of study systems, as this would only require the availability of plant and bird community data for a mosaic of habitat patches. Applying the framework to real-world data can yield a quantitative understanding of plant functional connectivity in different types of landscapes and ecosystems, as well as help to provide practical guidance to forest restoration projects."
} | 5,803 |
25434843 | PMC4248267 | pmc | 6,196 | {
"abstract": "Patterned structures of flexible, stretchable, electrically conductive materials on soft substrates could lead to novel electronic devices with unique mechanical properties allowing them to bend, fold, stretch or conform to their environment. For the last decade, research on improving the stretchability of circuits on elastomeric substrates has made significant progresses but designing printed circuit assemblies on elastomers remains challenging. Here we present a simple, cost-effective, cleanroom-free process to produce large scale soft electronic hardware where standard surface-mounted electrical components were directly bonded onto all-elastomeric printed circuit boards, or soft PCBs. Ag-PDMS tracks were stencil printed onto a PDMS substrate and soft PCBs were made by bonding the top and bottom layers together and filling punched holes with Ag-PDMS to create vias. Silver epoxy was used to bond commercial electrical components and no mechanical failure was observed after hundreds of stretching cycles. We also demonstrate the fabrication of a stretchable clock generator.",
"conclusion": "Conclusions The proposed method integrates important standard PCB design features like straight traces, vias, solderability, connectivity with standard hardware and can be used to design soft and stretchable PCBs the same way rigid or flexible PCBs are designed. Stencil or screen printing Ag-PDMS is a simple solution for large scale production of PCBs at low costs and the method of bonding them together and making vias for double sided PCBs is convenient and similar to current industrial processes. Double-sided soft PCBs had in average a low ohmic resistance of 2 Ohm/cm. Commercial electrical components can be bonded onto Ag-PDMS tracks using a standard Ag-epoxy to fabricate soft circuits. These circuits can be interfaced to rigid electronics using commercial ZIF connectors. An astable circuit including all these features was made to demonstrate the presented method. This technique also enables the fabrication of thin, soft and stretchable conductive leads for multielectrode arrays with mechanical properties closer to tissue than state-of-the-art polyimide-based neural interfaces. Such silicone-based implants can be used to stimulate or record from the brain or the spinal cord without damaging the delicate neural tissue even when implanted in the subdural region. This technology paves the way for a new generation of neuroprosthetic devices.",
"discussion": "Results and discussion Fabrication of soft PCBs using stencil printing and screen printing Stencil printing Large-area Ag-PDMS structures were fabricated using standard industrial printing techniques: stencil printing and screen printing (see Methods). Custom-made copper stencils were used as shadow masks for patterning structures of Ag-PDMS as shown in Fig. 1a . The adhesion between the PDMS substrate and the stencil was affected by the roughness of the copper surface. Stencils with smooth surface (no treatment) adhered strongly to the substrate and did not move during printing whereas thinner stencils produced by wet etching from both sides poorly adhered to the PDMS due to their rougher surface. Hence, thinner stencils were produced by wet etching one side of the copper stencil while protecting the other side with tape. 150 µm wide lines with 100 µm spacing were achieved using 100 µm thick stencils as shown in Fig. 1b . Such stencils were robust enough to be reused several times without getting deformed contrary to thinner stencils that were more fragile. Hexane was used as a cleaner between consecutive printings. Printing could be repeated several times using the same stencil without losing quality. Mishandled stencils tended to buckle and did not lay perfectly flat during printing which led to defects and unwanted short circuits between lines. Figure 1c shows the cross sections of lines printed using Ag-PDMS with different viscosity. High resolution patterns could not be achieved using Ag-PDMS with filler content below 22vol% since the material was flowing after the stencil was removed due to its lower viscosity. Prolonged use of Ag-paste during repeated printings accelerated the cross linking and aging of the composite thus increasing excessively the viscosity of the paste and introducing short circuits between lines. However, Ag-PDMS pastes could be stored at −24°C over a year without dramatic loss of performance. Figure 1f shows typical short circuits between lines resulting from bad printing. Such defects could be manually removed before the composite was cured or after curing by cutting away the undesired parts, as it is done with standard PCBs, and filling the cavities with uncured PDMS. Closed lines could not be printed using this method because there was no frame to hold the stencil together. Screen printing This was not anymore a problem when using screen printing since the screens are composed of a mesh on which a thick resin is applied. The screen was fixed on a frame and its vertical position could be adjusted with screws. The distance between the screen and the PDMS was set to 4 mm to prevent the resin to stick to the PDMS during printing. Hence a metallic mesh was preferred to a plastic mesh because of its superior rigidity to avoid buckling after such a large deformation. Large patterns could successfully be printed on 8″ wafers covered with PDMS as shown in Fig. 1d . Line widths of 70 µm and spacing of 50 µm were achieved as shown in Fig. 1e . Lines were 40 µm thick, which corresponds to the thickness of the resin on the screen. Smaller features could not be fully transferred and looked like dashed lines. Cleaning the mesh before each printing was necessary to remove residues clogging the mesh apertures. Figure 1g shows large soft PCBs produced using screen printing. The PCBs were soft enough to conform to various surfaces. Patterning conductive PDMS on elastomeric substrates have been investigated in quite a few ways by microcontact printing 20 , trench filling 21 or photocrosslinking conductive PDMS 22 . High resolution patterns of highly viscous pastes can be achieved using trench filling or photopatternable conductive PDMS but are challenging using microcontact printing. stencil printing and screen printing are commonly used in the electronics manufacturing process to print electronic circuits. Although large stencil printed patterns of Ag-PDMS were already reported by others 23 , we investigated here the feasibility of printing stretchable circuits of dimensions comparable to standard electronics. We also showed that screen printing was compatible with this material and that clogged Ag-PDMS can be removed to allow multiple printing. Electro-mechanical properties of Ag-PDMS composite Percolation in composite materials depends on the filler material, aspect ratio, quality of dispersion and matrix material and influences both electrical and mechanical properties. Conductivity and contact resistance were measured using Kelvin sensing on stencil printed Ag-PDMS stripes (40 mm × 5 mm × 0.1 mm) with different filler volume fractions ranging from 12% up to 25%. Cured samples were peeled off, placed on a glass slide and four probes were pressed against the sample using magnets. The volume content of Ag particles determines the size of percolation networks in the PDMS matrix and the conductivity of the composite. Electrical conductivity of 1 S/cm was measured in samples with 13vol% and then rapidly increased to 100 S/cm when adding small amounts of silver and finally reached 600 S/cm after loading 25vol% of silver in the PDMS matrix. The contact resistance followed the inverse behavior as shown in Fig. 2a . The data were best fitted with the following percolation model (red line) σ c = σ 0 ( c − c t ) t where σ 0 was 18168 S/cm, c t was 12.6% and t was 1.68. The changes in resistance R/R 0 under quasi-static uniaxial strain were measured similarly in a tensile test machine at the rate of 1%/min. Rapid increase in resistance occurred in samples with low silver content when samples with higher concentrations were stretched above 100% as shown in Fig. 2b . Samples with 23vol%, 24vol% and 25vol% were still conductive before rupture. Large dog-bone samples of Ag-PDMS 13vol%, 16vol%, 19vol%, 22vol%, 25vol% and pure PDMS were molded and mounted in the tensile test machine to measure its mechanical properties. Figure 2c shows stress strain curves for a strain rate of 0.1 mm/s. The elastic moduli were defined as the slope for the first 1% and are plotted against the silver content in Figure 2d . The large error bar for 25vol% can be explained by the outlying behavior of one of the three samples that showed higher stiffness and early fracture. The max strains at break are shown in Figure 2e . It appears that samples loaded with silver particles exhibited a higher strain at break that increased with the silver volume content and was maximal for 19%. Adding more silver seemed to fragilize the material and decrease the maximal elongation at rupture. Excessive amount of silver also increased the viscosity, thus making the printing difficult and the samples more brittle. To investigate the changes in Poisson ratio with silver content, Ag-PDMS dog-bone samples were fixed on a custom-made manual stretcher placed under a measurescope. Changes in width were measured while stretching uniaxially. The Poisson ratio was lower for composites with higher filling contents and decreased with applied strains as shown in Fig. 2f . Elastomers like PDMS are generally considered to be incompressible with a value of Poisson ratio of 0.5. Increasing the amount of fillers decreased the Poisson ratio that was shown to be strain dependent probably due to dewetting and vacuole formation 24 due to weak bonding between the silver particles and PDMS. Under tensile strain the volume of the composite increased, which decreased the volume content of Ag towards the percolation threshold, which explains the rapid increase in resistance. The influence of sample sizes on conductivity and stretchability was also investigated. Narrower tracks (2 mm, 1 mm, 0.6 mm, 0.3 mm, 0.25 mm, 0.2 mm and 0.15 mm) were stencil printed on the same substrate, cured, peeled off and placed on a glass slide. Figure 3a and 3b show resp. the max current densities and sheet resistances as a function of the line width. The sheet resistances unexpectedly decreased when reducing the track width, which is in contradiction with percolation theory. The electrical resistance of a composite conductor should increase when its dimensions become comparable to the size of the filler particles because of the lower probability of finding conductive pathways. The increase in maximum current density is consistent with the decrease in sheet resistance. The mean conductivity for each line width was plotted against the volume fraction in Fig. 3c (without error bars for clarity) and the data were fitted using the previously introduced percolation model with the same values of c t ant t . The fitted apparent conductivity significantly increased when the track width was decreased below 0.5 mm as shown in Fig 3d . These results suggested that the conductivity of the material was influenced by the size of the printed patterns. There are at least two possible explanations to this effect. The particles may come in closer contact with each other thus lowering the resistance at the interface because of higher compressive forces when printed through a narrower mask or an increase in particles concentration due to the lateral flow of PDMS after printing. The samples of the first batch of Ag-PDMS 25vol% with different lines widths were stretched to 50% at a speed of 1 mm/s and the change in resistance was plotted in Fig. 3e . The narrower lines showed less increase in resistance, which is consistent with their previously described superior performances. Standard PCBs often have copper traces of 200 µm in width. These results suggest that printing stretchable conductive circuit boards with track widths similar to that of rigid PCBs is feasible. For the production of double-sided soft PCBs, the top and bottom layers were bonded together and vias were created by punching holes through the board and filling them with the same Ag-PDMS paste as shown in Fig. 3f . Another method for creating through silicone vias reported elsewhere 23 consisted in bonding patterned layers of Ag-PDMS to Ag-PDMS vias printed onto a PMMA substrate and filling the gap with uncured PDMS using a syringe. Here, we aimed at providing a method similar to what is found in the industry where drilled holes are filled with a conductive paste. This approach was also successfully used with polyurethane composites for flexible electronic applications 25 . Bonding components and interfacing soft electronics with hard electronics Interconnecting electrical circuits can be done in a reversible manner using mechanical clamping or in an irreversible way using solder bonding. Zero Insertion Force (ZIF) connectors were used to interconnect soft PCBs to rigid standard PCBs. A stretchable ribbon cable with 8 leads was produced using stencil printing and was clamped between two ZIF connectors as shown in Fig. 4a . The ZIF connectors provided good electrical contacts even when the ribbon cable underwent large strains as high as 40% (see Supplementary Movie 1 ) but mechanically damaged the printed tracks after repeated manual stretching cycles due shear stress exerted by the metallic contacts on the soft Ag-PDMS leads. To solve this problem, the local stress can be delocalized by reinforcing the terminals with a sheet of polyimide or additional clamping on the PDMS. Novel designs of ZIF connectors including a second clamping system could significantly improve the reliability of the connector. Another way of interconnecting was to use Ag epoxy to bond the soft ribbon cable onto a rigid double-sided PCB. Figure 4b shows the interconnection between a stretchable ribbon cable with 12 conductors and a miniature custom-made connector. This solution allowed for smaller contact area and miniaturized interconnections but the bond is permanent. The Ag epoxy bonding technique was also used to mount SMD components onto the soft PCB. Chip resistors of various sizes were bonded between two tracks with different widths. Figure 4c shows 0406, 0603 and 0805 chip resistors after bonding. The samples were stretched to 20% over a thousand of cycles several cycles at 1 mm/s and the bond did not fail mechanically. When chip resistors were manually removed from the circuit by pulling on them some Ag-PDMS came off with the components suggesting good adhesion. High yield of the bonding was demonstrated with 6 × 7 arrays of LEDs = 84 contacts (see Fig. 4d ). A soft astable circuit generating a 2 kHz clock was produced using the described method and is visible in Fig. 4e . Finally, a stretchable clock generator assembled on a double-sided PCB including vias with LEDs flashing every second and a ZIF connector is showed in Fig. 4f . The frequency of the clock determined by the values of the resistor and capacitor remained stable during manual bending and stretching (see Supplementary Movies 2 and 3 )."
} | 3,820 |
22742196 | null | s2 | 6,197 | {
"abstract": "Bacteria isolated from marine sponges, including the Silicibacter-Ruegeria (SR) subgroup of the Roseobacter clade, produce N-acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge-associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI and sscI (sponge-associated symbiont locus A, B and C, luxR or luxI homologue). SsaI produced predominantly long-chain 3-oxo-AHLs and both SsbI and SscI specified 3-OH-AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild-type KLH11. AHLs and the ssaI transcript were detected in M. laxissima extracts, suggesting that AHL signalling contributes to the decision between motility and sessility and that it may also facilitate acclimation to different environments that include the sponge host."
} | 341 |
27540075 | PMC4991720 | pmc | 6,200 | {
"abstract": "To address the metabolic potential of symbiotic Aquimarina spp., we report here the genome sequence of Aquimarina sp. strain EL33, a bacterium isolated from the gorgonian coral Eunicella labiata . This first-described (to our knowledge) animal-associated Aquimarina genome possesses a sophisticated repertoire of genes involved in drug/antibiotic resistance and biosynthesis."
} | 95 |
30755173 | PMC6373157 | pmc | 6,201 | {
"abstract": "Background Methanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain. Results We integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden–Meyerhof–Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH. Conclusions This study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries. Electronic supplementary material The online version of this article (10.1186/s12864-019-5487-6) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions In conclusion, we have presented genomic and transcriptomic analyses of an industrially promising obligate methanotroph, Methylomonas sp. DH-1. The strain was grown on methane and methanol to analyze the shift of metabolism affecting by selection of substrates (Figs. 1 , 2 ). While some metabolic functions had been reported in previous studies, several novel functions were identified and characterized in this strain. Methylomonas sp. DH-1 possesses the active EMP pathway which main route for C1 assimilation in this strain. In addition, Methylomonas sp. DH-1 also operates a complete oxidative TCA cycle. Along with the existence complete serine cycle, these pathways may function in C1 assimilation and energy production. We also identified a flux shift of metabolism towards formaldehyde oxidation pathway, serine and TCA cycle in Methylomonas sp. DH-1 when substrate was changed from methane and methanol. Furthermore, a significant upregulation of carotenoid and hopanoid biosynthesis pathways under methanol growth might explain the resistance to high methanol concentrations observed in Methylomonas sp. DH-1. It appears that methanotrophs are very dynamic to respond to change of environmental parameters.",
"discussion": "Discussion In order to provide knowledge for methanotroph-based refineries, multi-omics can be used to define which metabolic pathways are active in certain conditions, and how cells response and adapt to new environments. In our previous work, the complete genome sequence of the newly isolated methanotroph Methylomonas sp. DH-1 was reported [ 19 ]. In the present study, a comprehensive characterization of the complete transcriptome of Methylomonas sp. DH-1 was provided and analyzed for the first time by an RNA-seq approach. This study provides in-depth knowledge about the metabolic pathways of this strain and reveals key differences in the transcriptional responses for certain metabolic pathways during growth in methane and methanol. In the well-characterized methanotrophs, pmo is expressed at the highest level for cultures grown on methane [ 14 – 17 ]. A previous study has determined that transcripts of pmoA are very stable, with a half-life in the range of hours to days [ 53 ] which supported the hypothesis that the higher expression levels of pmo compared to other enzymes in the C1 oxidation pathway led to the first step of oxidizing methane is relatively slower compared to subsequent steps. During growth on methanol, the pmo operon was dramatically downregulated, likely because pmo genes are not involved in oxidizing methane. This is consistent with our previous study in which MMO activity has been dropped more than 3-fold when DH-1 growth on methanol [ 11 ]. Methane therefore may be a key regulator for the expression of the pmo operon. Similar to that of M. trichosporium OB3b, a type II model methanotroph, the expression of pmo and smo strongly affected by selection of different substrates [ 54 ]. The expression level of pmo and smo and their activity extremely dropped when the growth was shifted from methane to methanol [ 54 ]. xoxF , Ln 3+ − dependent methanol dehydrogenase, has an important enzyme in methylotrophy, providing a new outlook on the distribution of methylotrophy in the bacterial community [ 55 ]. Interestingly, xoxF showed highly expression level without presence of Ln 3+ and the similar expression pattern of xoxF and pmo supported the assumption that xoxF could contribute to the methane oxidation process in Methylomonas sp. DH-1. In agreement with our hypothesis, in most recent, the structure and function of xoxF in M. buryatense 5GB1C has been reported by investigating the possibility of interaction between pMMO and XoxF [ 56 ]. The results indicated a XoxF monomer may bind to pMMO and suggested an alternative structure of MDH-pMMO association. On the other hand, M. trichosporium OB3b showed very low expression level of xoxF1 and xoxF2 in methane and methanol [ 54 ]. Furthermore, the expression level of xoxF1 , xoxF2 as well as mxaF in M. trichosporium OB3b were decreased when grown on methanol with the presence of 10 μM copper, highlighting the differences in gene expression regulation in response to the type of carbon sources available between Methylomonas sp. DH-1 and M. trichosporium OB3b. It should be noted that while M. trichosporium OB3b exhibited the “copper-switch” to control the expression of alternative forms of methane monooxygenase, the “copper-switch” was not exist in Methylomonas sp. DH-1. The discovery of typical type II methanotrophs metabolic pathways, such as the H 4 MPT pathway, H 4 F pathway, and complete serine cycle, in Methylomonas sp. DH-1 raised questions about the roles of these pathways in the central metabolism of this strain. From a previously published genome-scale model of M. buryatense 5GB1, a minor carbon flux is predicted via the H 4 MPT and H 4 F pathways [ 17 , 18 , 57 ]. However, these pathways were more active during growth on methanol, suggesting the improvement of carbon flux towards these pathway. This observation supports our hypothesis that the H 4 MPT and H 4 F pathways are mainly responsible for formaldehyde oxidation and contribute to carbon conversion via the serine cycle when grown on methanol. A partial serine cycle without ppc has been determined in various type I methanotrophs such as M. buryatense 5GB1 and M. alcaliphilum 20Z R which contributed a minor flux during growth in methane [ 17 , 18 ]. Likewise, the complete gene set implementing the serine cycle in Methylomonas sp. DH-1 should allow the minor carbon flux needed to produce acetyl-coA. In the type II methanotroph M. trichosporium OB3b, which typically uses the serine cycle as a main pathway for C1 assimilation, there are two kinds of ppc gene: ppc1 belongs to the non-regulated group and ppc2 belongs to the regulated group [ 14 ]. The existence of two functionally identical but different regulation systems in M. trichosporium OB3b allows control of flux through phosphoenolpyruvate-oxaloacetate in response to the serine cycle, and this flux is never blocked completely [ 14 ]. The presence of only regulated ppc in Methylomonas sp. DH-1 indicates that carbon flux through the serine cycle can be blocked in the absence of effectors. During culture on methanol, expression of ppc was strongly downregulated, possibly because metabolite effectors which activate ppc expression were absent. The growth rate of Methylomonas sp. DH-1 in methanol was significantly decreased, perhaps because carbon flux via the serine cycle may have been blocked under methanol growth. However, most of the genes in the serine cycle were upregulated in methanol, suggesting significant shifts occur in C1 assimilation pathways, from RuMP to serine cycle. Along with RuMP cycle, the serine cycle also could take the role of producing acetyl-coA. EMP is main variant of RuMP pathway which play major role for C1 assimilation to produce NADH and ATP in type I methanotrophs [ 16 – 18 ]. The shifts decrease flux towards EMP pathway which subsequently decrease ATP production. Instead, the available electrons from methanol oxidation, which not used for methane oxidation under methanol growth, are transferred to the electron transport chain follow by producing ATP via oxidative phosphorylation. In order to determine the detailed rearrangement of metabolic network involved methanol-grown, 13 C tracer analysis and constraint-based analysis of genome-scale metabolic network studies are needed. Thus, even the exist of the complete serine cycle in Methylomonas sp. DH-1 could not be main pathway for C1 assimilation, it could contribute to the control of carbon flux when shifting carbon substrates. One unsolved question surrounding the central metabolism of type I methanotrophs is whether the oxidative TCA cycle is complete. In the recent time, a complete oxidative TCA cycle has been demonstrated to operate in M. buryatense 5GB1, and it has showed three separate pathways for converting 2-oxoglutarate to succinyl-CoA [ 33 ]. In another study, highly branched TCA cycle at the 2-oxoglutarate node also has been reported in M. alcaliphilum 20Z R [ 18 ]. In this study, we also suggested Methylomonas sp. DH-1 possesses an complete oxidative TCA cycle. However, genomic analysis indicated at 2-oxoglutarate node, Methylomonas sp. DH-1 possesses 2-oxoglutarate dehydrogenase complex only but not 2-oxoglutarate ferredoxin oxidoreductase, succinate semialdehyde dehydrogenase or 2-oxoglutarate decarboxylase. Thus, the presence of highly branched TCA cycle in DH-1 remains to be elucidate. In addition, it seems that carbon flux though TCA cycle was reduced on methanol growth and the critical function of TCA under methanol growth has changed. In methanol-grown cells, TCA cycle mostly provide precursors for de novo synthesis but not reducing power such as NADH. Instead, it appears that the activation of formaldehyde oxidation in methanol growth could produce NADH. In our previous study, the carotenoid biosynthesis pathways which derived from MEP pathway has been proposed [ 19 ]. The dxs is the first and one of the most important rate-limiting step in the MEP pathway, and overexpression of dxs could improve the production of several downstream secondary metabolites such as isoprenoid and carotenoid [ 58 – 61 ]. The flux shift occurred to MEP pathway via the strong upregulation of two dxs homologs ( dxs1 and dxs2 ) led to the accumulation of carotenoids in methanol-grown cultures. Meanwhile, the extremely upregulation of hopanoid biosynthesis pathway might related to membrane modifications under methanol growth (Fig. 2 ). The function of hopanoids has been characterized in several organisms, including methylotrophic bacteria [ 62 , 63 ]. A lack of hopanoid biosynthesis increases sensitivity against toxins and osmotic stress. During growth on single-carbon compounds, methanol is generally converted to formaldehyde in the periplasm, and the formaldehyde is then transported and utilized in the cytoplasm. Given the toxic intermediates in this process, elevated maintenance of the inner and outer membranes is necessary. The role of hopanoids in maintaining membrane robustness and membrane barrier function is likely conserved across bacterial lineages. This function is possibly mediated through an interaction with lipid A in the outer membrane of Methylobacterium extorquens DM4 [ 63 ]. In addition, membrane function in the hopanoid-free Methylobacterium extorquens PA1 was lower [ 62 ]. Further investigation on the function of hopanoid biosynthesis pathway in property membranes of Methylomonas sp. DH-1 is needed to solve the question if hopanoid biosynthesis pathway could enable resistance to high methanol concentrations in Methylomonas sp. DH-1. Under methanol growth, the upregulation of carotenoid biosynthesis pathway, which produced pigmented carotenoid as antioxidant, and many regulatory defense systems against oxidative stress via damage repair and protection systems have been observed. It is speculated that such changes of these gene expression were induced by methanol which might induces ROS in Methylomonas sp. DH-1. A high expression of MEP pathway genes and an accumulation of carotenoids under stress conditions also describe previously reported in Haematococcus pluvialis [ 64 ]. That such speculation must be more rigorously confirmed by apply a system biology approach to reconstruct genome-wide of OxyR , SoxR , and SoxS regulatory networks under oxidative stress condition in methanotrophs."
} | 3,589 |
29167757 | PMC5686429 | pmc | 6,202 | {
"abstract": "Graphical abstract",
"conclusion": "6 Conclusion C. sticklandii has a great concern of utilizing proteins for producing renewable alternatives to the petroleum-based chemicals and fuels using a metabolic model at the genome-scale. Target genes for guiding metabolic engineering and novel pathways to be engineered have to be elucidated to the scientific community for competent biofuel production at industrial scale. Genome-scale model of C. sticklandii would provide a skeleton for predicting its metabolic behavior and emphasizing metabolic connections to biofuel synthesis. It will afford a guide for parallel reconstruction of genome-scale metabolic networks of related species. Of particular importance is the development of a regulatory circuit that allows a recombinant pathway to become part of a heterologous metabolic network for biofuel production [124] and tolerance [125] . Evolving genetic regulatory circuits would perform a self-regulating gene expression and enzyme activity in response to the precursor and or substrate supply in the host cell [126] , [127] . Genome-scale model of C. sticklandii will also provide a clue to design a synthetic pathway that needs to be integrated into the metabolic network of heterologous host in order to achieve optimal production levels. Optimized bioprocess parameters would be implemented for biofuel production from waste recycling of protein industry-based biomass in the biotechnology industry with eco-friendly manner. The present review concluded that model-driven systems metabolic engineering approach would advance the broaden applicability of using C. sticklandii for biofuel production in the industrial sector.",
"introduction": "1 Introduction Chemical and pharmaceutical industries are manufacturing fine chemicals in use of organic solvents and acids. Acetone is a solvent in the production of cordite, a smokeless ammunition propellant. Ethanol is a gasoline additive to increase octane and improve vehicle emissions. Butanol is used as an alternative liquid transportation fuel and can be catalytically converted into jet fuels. Moreover, butanol has proved to be a better biofuel than acclaimed ethanol owing to it is less corrosive and easily blend with gasoline [1] . Isobutyric acid and n -butyric acid are commercially used in the production of artificial fibers, plastics and herbicides. Isovaleric acid is mainly used for perfumery production and intensive care medicine [2] . The worldwide biofuel production reached 105 billion liters in 2010 and a contribution largely made up of ethanol and biodiesel [3] ( Fig. 1 ). Using biotechnological processes and renewable resources, the worldwide production of acetic acid exceeds 2 million metric tons per year. The worldwide annual production quantity of propionic acid was estimated to 377,000 t in 2006 [2] . Economic development of renewable chemicals and biofuel technologies rests on several important parameters. The social, environmental and technical crises and substrate availability are being the most important factors in determining bioprocess viability and economical feasibility [1] . Fig. 1 International Energy Statistics for global biofuel production and consumption. Fig. 1 Microorganisms are rich sources for natural products, some of which are used as fuels, commodity chemicals, fine chemicals, and polymers [4] . Genetically engineered microbial strains should have a metabolic capability of utilizing multiple substrates with variable composition and without catabolite repression, which are crucial for the development of economic processes [5] . A microbial strain that has diverse substrate utilization potential would offer a major competitive advantage to the biofuel production from various feedstock such as carbohydrates, lipids and proteins. 1.1 Carbohydrate-based substrates Acetone–butanol–ethanol (ABE) fermentation is a known metabolic process of Clostridium sp. for the production of acetone, n -butanol, and ethanol from lignocellulosic biomass degradation [1] , [6] , [7] . Starches and simple sugars derived from sugar cane and corn are the most commonly used feedstock for the industrial production of biofuels. Microbial fermentation converts sugars produced from lignocellulosic biomass into biofuels or biorefineries using Clostridium sp. The theoretical maximum calculated to be 0.939 mol butanol/1 mol glucose by ABE fermentation [8] and ∼1.33 mol butanol/1 mol glucose by mixotrophic fermentation [9] . Clostridium acetobutylicum has been studied as a model organism of biofuel production for several decades [10] . C. acetobutylicum ATCC 824 and C. beijerinckii BA101 were proved to increase n -butanol tolerance and for n -butanol production from carbohydrates [6] , [7] . Butanol production was previously commercialized from molasses produced from sugar cane industry using C. acetobutylicum \n [11] . C. beijerinckii BA101 mutant produced the highest concentration of n -butanol (17–21 g/L) from glucose across all microorganisms [12] . However, lignocellulosic biomass has to be pretreated for biofuel production under harsh conditions that requires a large amount of energy consumption [13] , [14] . 1.2 Lipid-based substrates Compared to cellulosic biomass, food waste holds several significant advantages to produce butanol, since it comprises with significant quantities of sugar, starch, fatty acids, proteins and minerals. The considerable amounts of these functionalized substrates can act as nutrients to proliferate the culture growth during ABE fermentation [15] , [16] . Even if lipids used for biodiesel generation using transesterification process, current production technology is not being economical to make biofuels through lipid fermentation [17] . However, a coproduct derived from high-value fatty acids could potentially make the biorefinery process economical [18] . The maximum theoretical yield of ethanol obtained from palmitic acid (1.38 g ethanol/g palmitic acid) is significantly higher than that obtained from glucose (0.51 g ethanol/g glucose) [19] , [20] . Clomburg and Gonzalez [19] calculated the maximum theoretical yield of 1 mol each of ethanol and hydrogen per mole of glycerol fermented with an ethanol production of 4.6 mmol/L/h. 1.3 Protein-based substrates Bioconversion process for releasing protein/amino acids from algal biomass or protein-rich waste may be easier than breaking down lignocellulosic to fermentable sugars [21] . The conversion efficiency of total sugar in food waste was up to 88%, but of protein was 40–70% [22] . Therefore, the conversion of proteins into biofuel is usually the rate limiting step during acidogenesis and solventogenesis phases [23] . Amino acids released by proteolysis of a protein-based substrate are catabolized to produce keto acids, which are synthesized into biofuels (ethanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol) [24] . Valine, leucine, threonine and isoleucine biosynthetic pathways have been overexpresed in Escherichia coli host for the production of isobutanol [25] , 2-methyl-1-butanol [26] , and 3-methyl-1-butanol [27] . Engineered E. coli achieved a yield of ∼4 g/l of alcohols from a yeast extract containing 21.6 g/l of amino acids at 56% of the theoretical yield [28] . Rerouting nitrogen flux in E. coli was allowed to producing up to 4,035 mg/L of alcohols from Saccharomyces cerevisiae , E. coli , Bacillus subtilis and microalgae that can be used as protein sources containing ∼22 g/l of amino acids [21] . E. coli carrying Clostridial butanol dehydrogenase ( bdh ) gene metabolically engineered for the conversion of protein hydrolysate to n -butanol and n -pentanol [28] . Molecular and biochemical characteristics of many Clostridium species have been extensively studied for biofuel production. However, industrial and economic importance of them on the protein-based waste are hindered due to a lack of knowledge of their complex nature of metabolic and regulatory networks at a genome-scale. Hence, systems-metabolic engineering of industrially important Clostridium would advance in the development of economically viable processes."
} | 2,045 |
28699345 | PMC5548412 | pmc | 6,206 | {
"abstract": "Because\nof their inherent rigidity and brittleness, inorganic materials have\nseen limited use in flexible thermoelectric applications. On the other\nhand, for high output power density and stability, the use of inorganic\nmaterials is required. Here, we demonstrate a concept of fully inorganic\nflexible thermoelectric thin films with Ca 3 Co 4 O 9 -on-mica. Ca 3 Co 4 O 9 is\npromising not only because of its high Seebeck coefficient and good\nelectrical conductivity but also because of the abundance, low cost,\nand nontoxicity of its constituent raw materials. We show a promising\nnanostructural tailoring approach to induce flexibility in inorganic\nthin-film materials, achieving flexibility in nanostructured Ca 3 Co 4 O 9 thin films. The films were grown\nby thermally induced phase transformation from CaO–CoO thin\nfilms deposited by reactive rf-magnetron cosputtering from metallic\ntargets of Ca and Co to the final phase of Ca 3 Co 4 O 9 on a mica substrate. The pattern of nanostructural\nevolution during the solid-state phase transformation is determined\nby the surface energy and strain energy contributions, whereas different\ndistributions of CaO and CoO phases in the as-deposited films promote\ndifferent nanostructuring during the phase transformation. Another\ninteresting fact is that the Ca 3 Co 4 O 9 film is transferable onto an arbitrary flexible platform from the\nparent mica substrate by etch-free dry transfer. The highest thermoelectric\npower factor obtained is above 1 × 10 –4 W m –1 K –2 in a wide temperature range,\nthus showing low-temperature applicability of this class of materials.",
"conclusion": "4 Conclusions A fully inorganic flexible film,\nCa 3 Co 4 O 9 -on-mica, has been developed.\nA nanostructural tailoring approach has been demonstrated to induce\nmechanical flexibility in Ca 3 Co 4 O 9 thin films. The nanostructured Ca 3 Co 4 O 9 film is obtained by thermally induced phase transformation\nfrom the CaO–CoO thin film deposited on the mica substrate\nby reactive rf-magnetron cosputtering to the final phase of Ca 3 Co 4 O 9 . Mica acts as a flexible substrate\nand at the same time as a sacrificial layer for the film transfer\nonto other flexible platforms. The nanostructure of the film is influenced\nby the initial arrangements of the CaO and CoO phases in the as-deposited\nfilms, which is controlled by controlling the deposition conditions:\ndeposition temperature and percentage of oxygen in the gas mixture.\nFlexible films are bendable to the bending radius of 14 mm without\nany deterioration of thermoelectric performance. The maximum power\nfactor of the flexible film is 1.18 × 10 –4 W\nm –1 K –2 near 300 °C and does\nnot change much as a function of temperature within the temperature\nrange measured. With this high power factor and mechanical flexibility,\nthe present films can be promising in the area of flexible thermoelectrics.\nFurther enhancement of the power factor is possible by optimal doping.\nThe present approach can also be applicable to grow flexible films\nof other compounds in layered cobaltate family.",
"introduction": "1 Introduction Microscale electronic\ncomponents tend to operate on battery power, 1 which has limitations on their lifetime and requirement for recharging.\nThis is not desired for wearable devices, where a possible solution\ncould be the scavenging of body heat for electrical power generation\nby flexible thermoelectric converters (TEC). 2 However, for wearable and other flexible applications, a technology\ntransformation is required from rigid thermoelectrics to flexible\nthermoelectrics. Organic materials, because of their inherent\nflexibility, have been preferred over inorganic materials for this\npurpose. Extensive investigations have been done on organic materials, 3 − 5 with high thermoelectric performance reported for the conjugated\npolymer, PEDOT:PSS, with a thermoelectric figure of merit, ZT , of 0.25. 6 Despite the advantages\nof low material cost and solution-synthesis possibility, polymer materials\ntypically have low output power density and stability. 7 , 8 For high output power density and reliable performance over longer\nperiod of time, particularly in hostile environments, the use of inorganic\nmaterials is inevitable. However, it then becomes necessary to overcome\nthe problem of material rigidity. Recently, there have been\nsome investigations on developing flexible TEC based on inorganic\nmaterials. 9 , 10 In these investigations, flexible platforms\nare used to hold the thermocouples of inorganic materials, and the\nlegs of the thermocouples are subjected to temperature gradient in\nan out-of-plane direction of the flexible platform. The disadvantage\nof such thermocouple arrangements (vertical arrangements), leg height\nof the thermocouples being in the micrometer range, is that the temperature\ngradient along the active materials is low, resulting in a low output\nvoltage from the modules. Further, maximum power output from a wearable\nthermoelectric\ndevice requires thermal matching between the body skin and air, and\nfor that 3–5 mm leg height is investigated to be appropriate. 11 Achieving such a leg height in a flexible module\nwith vertical leg arrangements is quite challenging. An alternative\noption can be the lateral arrangement of thermocouples, where the\nthickness of the leg materials is not important as they are subjected\nto temperature gradient along their length, in parallel with the substrate\nplane. 12 The additional advantage of such\narrangements is that a large number of thermocouples can be accommodated\nin a small area. However, with such lateral arrangements of thermocouples,\nboth the substrate and the thin leg materials need to be mechanically\nflexible. There have been some attempts for developing flexible\nthermoelectric devices with such lateral arrangements of thermocouples.\nFor that, thin legs of inorganic materials are deposited on flexible\npolymer substrates by the printing method, for example, screen printing,\ninkjet printing, and dispenser printing. 13 − 16 However, the problem with these\nprinting techniques is that the low processing temperature of the\nfilm, restricted by low-temperature sustainability of the polymer\nsubstrate, causes rough interfaces of the grains in the film, resulting\nin the scattering of charge carriers and thus a drastic reduction\nin the electrical conductivity. To reduce the grain boundary scattering\nof charge carriers, the thermocouple legs can be deposited by sputter-deposition\non flexible substrates. 17 , 18 However, the mechanical\nflexibility of the leg materials is still a challenge, which needs\nto be addressed by inducing mechanical flexibility in inorganic thin\nfilms but with no deterioration of their electronic properties. Recently,\nZhou et al. have developed carbon nanotube-based flexible TEC for\nroom-temperature wearable applications; 19 however, its applicability above room temperature has not been examined. Tailoring the structure on the nanoscale can induce new mechanical\nproperties in inorganic materials. For example, pristine Al 2 O 3 is rigid in nature, but hierarchical nanoarchitectures\nhave been reported to produce squeezable Al 2 O 3 , with 50% recoverability. 20 Nanostructural\nengineering has been used to tailor the electronic and phononic properties\nof inorganic thermoelectric materials for the enhancement of their\nthermoelectric efficiency. 21 − 25 However, such experiments to induce mechanical flexibility in these\nmaterials are unexplored. Here, we report the growth of flexible\nCa 3 Co 4 O 9 thin films on a flexible\nmica substrate. A novel nanostructural tailoring approach has been\ndemonstrated to induce flexibility in Ca 3 Co 4 O 9 thin films without significant effects on their electronic\nproperties. Flexible Ca 3 Co 4 O 9 films\ncan be applicable in a wide temperature range from room-temperature\nwearable applications to waste-heat recovery from hot curved surfaces\n(e.g., hot pipes) and for applications in hostile environments. Thermoelectric\nperformance of the investigated films has been evaluated in terms\nof their power factors. High power factor (= S 2 /ρ, where S is the Seebeck coefficient\nand ρ is the electrical resistivity) is more important than\nlow thermal conductivity to achieve a high output power, 26 in particular, for low-power applications, such\nas wearable applications. However, sustaining a high power factor\nin flexible materials comparable to their pristine bulk values is\nquite challenging. The formation of nanolaminar platelets is typical\nof Ca 3 Co 4 O 9 because of its inherently\nlayered structure. We show that the size and orientation of these\nplatelet-like grains can be controlled to achieve flexible mechanical\nproperties of the films without compromising with their thermoelectric\nperformance. The nanostructured Ca 3 Co 4 O 9 films are produced by thermally induced phase transformation\nfrom CaO–CoO thin films deposited on mica substrates by reactive\nrf-magnetron cosputtering from elemental targets of Ca and Co. Muscovite\nmica is chosen as the substrate as it can act as a flexible substrate\nand at the same time can sustain high processing temperature of 700\n°C. Muscovite mica forms a layered structure, where aluminosilicate\nlayers are loosely bound by the boundary layer\nof potassium (K + ) ions, which is bendable and easily cleaved\nalong the boundary layer. Further, the film is easily transferable\nfrom mica by dry transfer, that is, mica can also act as a sacrificial\nlayer for the transferable film.",
"discussion": "3 Results and Discussion 3.1 Structure of the Films Figure 1 a shows\nan optical image of an as-deposited film, which was deposited with\nno substrate heating, that is, the substrate was kept at room temperature\n20 °C during sputtering deposition, and hence, the film is denoted\n( T s : 20 °C). Similarly, a series\nof other films ( T s : 225 °C), ( T s : 375 °C), and ( T s : 675 °C) are named after their deposition temperatures\n225, 375, and 675 °C, respectively. The as-deposited film is\nyellow in color. This appearance is similar for the rest of the samples\n(not shown). The as-deposited films consist of CaO–CoO phases,\nwhich is consistent with the observations on a sapphire substrate. 26 Figure 1 b shows the postannealed film ( T s : 20 °C). After annealing, all samples turn dark, as shown in Figure 1 b. This change in\ncolor is attributed to the phase transformation from the CaO–CoO\nphase to the final phase of Ca 3 Co 4 O 9 . In our previous study, we demonstrated the occurrence of three-stage\nphase transformation during annealing, leading to the formation of\nthe final phase of Ca 3 Co 4 O 9 . 27 Figure 1 Optical image of (a) as-deposited CaO–CoO film,\n(b) annealed Ca 3 Co 4 O 9 film, and (c)\nXRD pattern\nof the postannealed film ( T s : 20 °C). Figure 1 c shows the θ–2θ XRD scan\nfor the postannealed film ( T s : 20 °C).\nBroadened peaks at around 2θ = 8.66°, 17.51°, 26.62°,\nand 35.85° occur from the (00 l ) planes of muscovite\nmica. Diffraction peaks at 2θ = 16.42°, 24.73°, and\n33.25° are observed, originating from the (002), (003), and (004)\nplanes of Ca 3 Co 4 O 9 . The peak from\nthe (001) plane of Ca 3 Co 4 O 9 is not\nvisible here as it coincides with the broadened peak of mica at 2θ\n= 8.66°. Apart from the (00 l ) planes of Ca 3 Co 4 O 9 , one low intense peak from the\n(−201) plane is visible in Figure 1 c, which indicates that the film ( T s : 20 °C) is not singly oriented, but it\nhas grains with a mixed orientation. The XRD peaks in the θ–2θ\nXRD scan of the annealed films ( T s : 225\n°C), ( T s : 375 °C), and ( T s : 675 °C) are so weak that they almost\ncoincide with the background (see Figure S-1 of Supporting Information). This is because the orientation of the\nCa 3 Co 4 O 9 film might not satisfy Bragg’s\ncondition in the out-of-plane direction, which is consistent with\nthe previous observation for the CaCo 4 O 9 film\ngrown on SrTiO 3 (111). 27 The\norientation and the crystal structure of the films were investigated\nby TEM and SEM and are discussed later. Figure 2 a–d shows the SEM images of the as-deposited\nfilms ( T s : 20 °C), ( T s : 225 °C), ( T s : 375\n°C), and ( T s : 675 °C), and Figure 2 e–h shows\nthe SEM images of the postannealed films. The morphology of the as-deposited\nfilms changes from sample to sample, which is attributed to the different\ndeposition temperatures of the films. The morphology of the annealed\nfilms also varies from sample to sample. The formation of platelet-like\ngrains in the postannealed films is evident from Figure 2 . Because of the inherently\nlayered structure, the formation of nanolaminated platelets is typical\nfor Ca 3 Co 4 O 9 . Controlling the size\nand orientation of these nanolaminar platelets is not trivial in the\nfilms. 29 − 32 Here, we have modified the orientation of the nanolaminated grains\nin the films independent of the substrate by controlling the growth\ncondition. Figure 2 a shows the SEM image of the annealed film ( T s : 20 °C), showing both the in-plane and out-of-plane\norientations of the nanolaminated grains of Ca 3 Co 4 O 9 . In the films ( T s : 225\n°C), ( T s : 375 °C), and ( T s : 675 °C), the nanolaminated grains tend\nto align nearly vertically (as shown in Figure 2 f–h), that is, the c -axis of the grains is along the in-plane direction of the sample.\nThe phase of these films has been confirmed by TEM analysis and is\ndiscussed later. The thickness of the nanolaminated grains in the\nfilm ( T s : 225 °C) is found not to\nbe uniform; certain distribution in the grain thickness is evident\nfrom the SEM image. The thickness of the nanolaminated grains in the\nsample ( T s : 375 °C) is found to be\nalmost uniform (also evident from Figure 2 g) and estimated to be around 50 nm. When\nthe deposition temperature is increased to 675 °C, a distribution\nin the grain thickness is observed in the film ( T s : 675 °C). SEM images of the larger area of all\nas-deposited and postannealed films are provided in Figures S-2 and S-3 in the Supporting Information. Figure 2 SEM images\nof (a) as-deposited film ( T s : 20 °C),\n(b) as-deposited film ( T s : 225 °C),\n(c) as-deposited film ( T s : 375 °C),\n(d) as-deposited film ( T s : 675 °C),\n(e) postannealed film ( T s : 20 °C),\n(f) postannealed film ( T s : 225 °C),\n(g) postannealed film ( T s : 375 °C),\nand (h) postannealed film ( T s : 675 °C). Different arrangements of the\nnanolaminated grains in the annealed films is due to the different\nself-arrangements of the grains during nanostructural evolution during\nphase transformation, which is likely to be influenced by the initial\narrangements of CaO and CoO phases in the as-deposited CaO–CoO\nfilms. The energetic constraints that guide the self-arrangements\nare anticipated to include the surface and interface energy minimizations,\nas well as strain energy minimization. The self-assembly growth of\nthe layered cobaltate in the chemical solution deposition (CSD) technique\nwas studied before. 28 The oriented growth\nof the films was explained as owing to the external stress due to\nsolvent evaporation. In another study, Fu et al. reported the c -orientation of the Ca 3 Co 4 O 9 film grown on a polycrystalline Al 2 O 3 substrate\nby the CSD technique. 33 They argued that\nthe interactive force of the (00 l ) plane of Ca 3 Co 4 O 9 with the Al 2 O 3 (00 l ) plane is stronger than that in other planes, 34 and hence, the Ca 3 Co 4 O 9 (00 l ) plane tends to nucleate onto the Al 2 O 3 (00 l ) plane serving as seeds\nfor c -axis-oriented growth, resulting in the c -axis self-assembled orientation. Therefore, the substrate\nis believed to have a stronger impact on the selection of the film\norientation. However, in our study, the various orientations of the\ngrains of polycrystalline Ca 3 Co 4 O 9 on the same substrate under different deposition conditions negate\nthe argument on substrate influence on the texture selection of the\nfilm. In our case, it is rather so that the distribution of crystallographic\norientations of the grains in a polycrystalline film evolves during\npostdeposition annealing through a number of kinetic processes. 35 The final texture of the film depends on which\ntexture-selection mechanism and driving force dominates. In the present\ncase, the different arrangements of CaO and CoO nanophases in the\nas-deposited films drive the strain force in different directions,\nleading to different nanostructures of the postannealed films. Figure 3 a shows a\ntypical cross-sectional TEM image of an annealed film ( T s : 675 °C). The near vertical orientation of the\nnanolaminated grains in Figure 3 a is consistent with the observation from the SEM image analyses.\nThe compositional analyses by EDS confirm the Ca to Co ratio to be\n≈ 0.73, which corresponds to the Ca/Co ratio of 0.75 in Ca 3 Co 4 O 9 . The presence of an amorphous\nlayer of thickness ∼50 nm between the substrate and the film\nis evident from Figure 3 a. The amorphous layer is formed because of the high-temperature\ntreatment during annealing. In the amorphous layer, the presence of\nCa (21.3 at %) along with the elements from the mica substrate, O\n(55.7 at %), Al (7.9 at %), Si (9.9 at %), K (1.0 at %), and Fe (4.2\nat %), is confirmed by EDS analyses, however, with no trace of Co.\nThe proportion of O, Al, Si, K, and Fe in the amorphous layer is found\nto be equivalent to that of the mica substrate. This indicates that\nat 700 °C, the layered structure of mica near the interfacial\nregion collapses, forming an amorphous layer through the absorption\nof Ca. The formation of such an interfacial layer was confirmed for\nall annealed films (not shown). Figure 3 b shows the top view of the TEM image of the annealed\nfilm ( T s : 675 °C). The grains are\nfound to form a closed network, which is consistent with the observation\nfrom the SEM analyses. Such a network formation is desirable for avoiding\nany disruption of transport of charge carriers during flexible applications.\nThe presence of void spaces between the grains is visible in Figure 3 b, which indicates\nthat the film ( T s : 675 °C) is not\n100% dense. This is consistent with the SEM observation in Figure 2 h. A high-resolution\nTEM image in the inset of Figure 3 b shows the lattice imaging of the nanolaminated grains.\nFrom the lattice imaging, the interlayer spacing ( d -spacing) of the layered cobaltate is confirmed to be around 10.7\nÅ, which matches with the d -spacing for Ca 3 Co 4 O 9 . From the TEM image analyses,\nit is clear that the nanolaminated grains are not perfectly vertically\naligned (see Figure 3 a), that is, the c -axis of the grains makes a certain\nangle of inclination (5°–25°) with the substrate\nplane. Because of such an out-of-plane alignment of the nanolaminated\ngrains, Bragg’s condition is not satisfied, and hence, the\nXRD peaks are weak in the θ–2θ XRD scan. Figure 3 (a) Cross-sectional\nTEM image of a typical postannealed sample ( T s : 675 °C). The amorphous layer is of Ca (21.3 at %),\nO (55.7 at %), Al (7.9 at %), Si (9.9 at %), K (1.0 at %), and Fe\n(4.2 at %). (b) TEM image of the postannealed sample ( T s : 675 °C) taken from the top of the film surface. 3.2 Flexibility\nand Transferability of the Film Figure 4 a shows a typical flexible film prepared\nfrom the sample ( T s : 675 °C). Figure 4 b shows the different\nsteps leading to the thin flexible film. In step 1, the film is attached\nto a glass slide by wax in an upside down position. In step 2, the\nthickness of the mica substrate is reduced to 100 μm by the\nphysical delamination process. To further reduce the substrate thickness,\nmica is delaminated by a sticky tape, as shown in step 3. After repeating\nsuch delamination several times, the substrate thickness is reduced\nto 20 μm, as shown in step 4. In step 5, the glass slide is\nkept in acetone for 12 h so that the wax is completely dissolved and\na thin flexible film can be isolated. The film is bendable to a bending\nradius of 14 mm without any deterioration of its physical properties. Figure 4 (a) Image\nof the thin flexible film ( T s : 675 °C).\n(b) Demonstration of the preparation of the thin film from the postannealed\nfilm ( T s : 675 °C). Figure 5 a shows a cross-sectional SEM image of the flexible film ( T s : 675 °C). The inset of Figure 5 a shows the magnified image\nof a small cross-sectional portion of the film. The substrate thickness\nis around 20 μm. Figure 5 b shows a magnified image of the interfacial region of the\nfilm. The average film thickness is 250 nm. The inset of Figure 5 b shows the magnified\nimage of a small portion of the film. The arrangement of the nanolaminated\ngrains of Ca 3 Co 4 O 9 is clearly visible\nin the image. Such grain arrangements enable the film ( T s : 675 °C) to withstand higher stress (tensile and\ncompressive stress) developed due to bending. The epitaxially grown\noriented Ca 3 Co 4 O 9 films do not allow\nsuch bending without developing cracks. Therefore, we grow polycrystalline\nfilms with nanolaminated grains with their c -axis\nrandomly oriented in the sample plane (i.e., standing basal planes).\nThis arrangement of the grains results in a network formation with\ngaps between the grains (as shown in Figure 3 b), which allow the relative motion and grain\nboundary/dislocation glide during bending, thus sustaining bending\nstress. In a fully dense oriented film, this relative motion is not\npossible and thus develops cracks to release the bending stress. Observation\nby an optical microscope confirms the absence of cracks in the film\neven after the repeated bending of the film to the bending radius\nof 14 mm in both directions. Figure 5 c shows an optical image of a small area (3.2 ×\n2.4 mm 2 ) of the film before and after bending. The film\nsurface before bending is seemingly flat; however, after bending,\nsome local curvatures in the film are developed from the compressive\nstress due to bending (evident from Figure 5 c) but with no crack on the surface of the\nbended film. Thus, it is confirmed that the film is able to sustain\nboth tensile stress and compressive stress when it is subjected to\na bending radius of 14 mm and thus has no effect on the thermoelectric\nproperties of the film (see section 3.3 for more details on thermoelectric properties).\nBecause of similar grain arrangements, the films ( T s : 225 °C) and ( T s : 375\n°C) have been found to withstand bending stress when they are\nsubjected to bending. Figure 5 (a) Cross-sectional SEM image of the flexible film ( T s : 675 °C), (b) magnified cross-sectional\nSEM image of the flexible film ( T s : 675\n°C) and (c) optical image of a small area of the film ( T s : 675 °C) before and after bending. Another promising aspect of the\npresent study is the transferability of the film to other flexible\nplatforms. With the emergence of flexible thermoelectrics, transfer\nof the films from the rigid substrate onto a flexible platform is\na major challenge. Strategies such as surface-energy-assisted transfer, 36 water-penetration-assisted mechanical transfer, 37 film transfer by using ultrasonic water bath, 38 and carrier-polymer-assisted transfer 39 have been demonstrated to transfer a monolayer\nor a few atomic layers of metal sulfide onto flexible polymer platforms;\nhowever, these strategies have not yet been examined on thick films\n(say thickness of several hundred nanometers). Recently, Lu et al.\nhave demonstrated the possibility of transfer of thick films by etching\nof sacrificial water-soluble layers. 40 In the present study, we examined if a 250 nm thick Ca 3 Co 4 O 9 film can be mechanically transferred\nfrom the parent mica substrate to another flexible platform. The transferability\nof the film was examined by transferring the film on a sticky tape.\nThe different steps of film transfer are shown in Figure 6 a. In step 1, one side of the\nfilm is marked by a sharp blade. In the second step, a tape was stuck\nto the film. After that, the film was isolated from the mica substrate\nby stripping (step 3). Figure 6 b shows the optical image of the back surface of the mechanically\nstripped film. Some leftover thin mica layers were still seen sticking\nto the back surface of the stripped film ( Figure 6 b). This, however, does not affect the functionality\nof the film as one exposed surface is sufficient for TEC. Figure 6 (a) Demonstration\nof the transformation of the film onto a sticky tape, (b) optical\nimage of the back surface of the stripped film, and (c) optical image\nof the small portion of the back surface of the stripped film. However, the functionality of\nthe film can be effected by the microcracks. Figure 6 c shows the optical image of a small area\n(3.2 × 2.4 mm 2 ) of the back surface of the stripped\nfilm. The presence of microcracks on the exposed part of the film\nis evident from Figure 6 c. To avoid microcracks, we instead remove the mica from the substrate\nside in a way similar to that demonstrated before in Figure 4 a, however, with one exception.\nThe film, instead of sticking to a glass slide, is adhered to a flat\nand sticky surface of a sticky tape (as shown in Figure 7 a). Figure 7 b shows the back surface of the film after\nthe removal of mica. Partial presence of the mica layers is still\nobserved as before, but no microcracks are observed by optical microscopic\nanalyses. Figure 7 c\nis a typical optical image of a small area (∼3.2 × 2.4\nmm 2 ) of the back surface of the film, which shows no cracks\non the film. Figure 7 (a) Optical image of the film ( T s : 675 °C) from the substrate side after the film is adhered\nto a sticky tape, (b) image of the film after the removal of mica\nfrom the substrate side, (c) optical image of the small portion of\nthe film after the removal of mica, and (d) image of the bended film\nafter it is transferred to the sticky tape. 3.3 Thermoelectric Properties Figure 8 a shows the temperature-dependent\nelectrical resistivity of all annealed films. The electrical resistivity\nof all films does not vary much as a function of temperature until\n250 °C. Beyond 250 °C, a rapid increase in the electrical\nresistivity is observed with temperature, which is attributed to the\nrelease of oxygen from the films. 41 − 44 The rate of increase in the electrical\nresistivity of all films is not the same. Above 250 °C, the ρ\nversus T curve of the film ( T s : 20 °C) is much steeper than those of other films. A\nnominal increase in the electrical resistivity with the temperature\nof the film ( T s : 675 °C) as compared\nto other films is likely due to the fact that it prevents the release\nof oxygen to a greater extent at high temperatures. The lower tendency\nof oxygen release from the film ( T s : 675\n°C) is attributed to its larger grain size than that of the film\n( T s : 375 °C) (see Figure 3 g,h for comparison). This is\nbecause the release of oxygen is more probable from the region near\nthe surface of the grains, and with the increase in the size of the\ngrains, the surface-to-volume ratio decreases, which in turn reduces\nthe effect of oxygen release. Note that the electrical resistivity\nmeasurement was performed in low-pressure helium gas, increasing the\ntendency of oxygen release at high temperatures. The oxygen release\nwill be very limited under an atmospheric condition. However, the\nmain focus of the present study is the low-temperature applicability\nof Ca 3 Co 4 O 9 films, particularly for\nwearable applications, and hence, high-temperature stability is of\nlimited importance. Near room temperature, the electrical resistivity\nof the films ( T s : 20 °C), ( T s : 225 °C), ( T s : 375 °C), and ( T s : 675 °C)\nis 29.73, 25.00, 20.30, and 16.46 mΩ cm, respectively. The highest\nelectrical resistivity of the film ( T s : 20 °C) is attributed to both out-of-plane and in-plane orientations\nof the grains in the film. Because of the inherently layered structure,\nthe physical properties of Ca 3 Co 4 O 9 are anisotropic in nature. The electrical resistivity along the c -direction of Ca 3 Co 4 O 9 is higher than that in the ( a , b ) plane. Because of both out-of-plane and in-plane orientations of\nthe grains, the resistivity of the film ( T s : 20 °C) is higher. The room-temperature value of the electrical\nresistivity of the film ( T s : 675 °C),\nalthough several times higher than textured Ca 3 Co 4 O 9 thin films, 45 is comparable\nwith the values obtained from undoped polycrystalline bulk Ca 3 Co 4 O 9 . 46 − 51 As previously mentioned, the vertical arrangement of the nanolaminated\ngrains of Ca 3 Co 4 O 9 is favorable for\nflexible applications. Repeated bending (100 times) of the film ( T s : 675 °C) shows no deterioration of electrical\nconductivity. It was the same for the films ( T s : 225 °C) and ( T s : 375 °C). Figure 8 Temperature-dependent\n(a) electrical resistivity, (b) Seebeck coefficient, and (c) power\nfactor of all films from room temperature to 400 °C before bending.\nComparison graph of (d) electrical resistivity, (e) Seebeck coefficient,\nand (f) power factor of the film ( T s :\n675 °C) before and after bending. Figure 8 b\nshows the temperature-dependent Seebeck coefficient ( S ) of all annealed films. Seebeck coefficient of all films varies\nwith temperature following the same manner as electrical resistivity.\nThe highest value of Seebeck coefficient near room temperature is\nobtained as 118 μV/K from the film ( T s : 675 °C). Near room temperature, not much variation of Seebeck\nvalues is observed for different films and remains within 111–118\nμV/K, which is comparable to the reported values for bulk Ca 3 Co 4 O 9 . 47 − 49 Below 150 °C, no\nconsiderable variation in the Seebeck coefficient is observed among\nthe films. Figure 8 c shows the power factor (PF = S 2 /ρ)\nof all films as a function of temperature. Near room temperature,\npower factors of ∼1 × 10 –4 W m –1 K –2 are obtained from the films ( T s : 675 °C), achieving the highest value of 1.18 ×\n10 –4 W m –1 K –2 near 300 °C. The power factor in the film ( T s : 675 °C), unlike the bulk sample, is nearly flattened\nwith temperature. It is remarkable for flexible oxide thin films to\nexhibit such a high power factor near room temperature. Liu et al.\ndemonstrated a free-standing Ca 3 Co 4 O 9 /PEDOT:PSS composite thin film; however, the room-temperature value\nof power factor was almost 7 times lower than the value obtained from\nthe flexible film ( T s : 675 °C). 52 Near room temperature, the power factor of the\nflexible film ( T s : 675 °C) is comparable\nto the values reported for undoped bulk polycrystalline Ca 3 Co 4 O 9 , 49 − 52 and further enhancement of power factor of the film\nis possible by optimal doping. 53 , 54 To examine the\nbending effect on the thermoelectric properties of the flexible film\n( T s : 675 °C), its Seebeck measurement\nwas performed after it was subjected to 100 times bending in both\ndirections; however, no notable change in the results is observed;\nwhatever variation in the Seebeck coefficient and electrical resistivity\nis found, it is well below the error limit specified by the ULVAC-RIKO\nZEM3 system. Figure 8 d–f compares the electrical resistivity, Seebeck coefficient,\nand power factor, respectively, of the film ( T s : 675 °C) before and after bending. No remarkable change\nin the values of the Seebeck coefficient, electrical resistivity,\nand power factor is observed. Small fluctuation in the values is well\nbelow the error limit. For flexible applications, the flexibility\nof the substrate is necessary but not sufficient; the film also needs\nto be flexible. Considering both the mechanical flexibility and thermoelectric\nproperties, the presently developed Ca 3 Co 4 O 9 films thus improve on other reports on flexible films."
} | 7,920 |
25620851 | PMC4297880 | pmc | 6,208 | {
"abstract": "Optimization of the production rate of biomass rich in N (e.g. for protein) or C (e.g. for biofuels) is key to making algae-based technology commercially viable. Creating the appropriate conditions to achieve this is a challenge; operational permutations are extensive, while geographical variations localise effective methods of cultivation when utilising natural illumination. As an aid to identifying suitable operational envelopes, a mechanistic acclimative model of microalgae growth is used for the first time to simulate production in virtual systems over a broad latitudinal range. Optimization of production is achieved through selection of strain characteristics, system optical depth, nutrient supply, and dilution regimes for different geographic and seasonal illumination profiles. Results reveal contrasting requirements for optimising biomass vs biofuels production. Trade-offs between maximising areal and volumetric production while conserving resources, plus hydrodynamic limits on reactor design, lead to quantifiable constraints for optimal operational permutations. Simulations show how selection of strains with a high maximum growth rate, U \n m , remains the prime factor enabling high productivity. Use of an f/2 growth medium with a culture dilution rate set at ~25 % of U \n m delivers sufficient nutrition for optimal biomass production. Further, sensitivity to the balance between areal and volumetric productivity leads to a well-defined critical depth at ~0.1 m at which areal biofuel production peaks with use of a low concentration f/4 growth medium combined with a dilution rate ~15 % of U \n m . Such analyses, and developments thereof, will aid in developing a decision support tool to enable more productive methods of cultivation. Electronic supplementary material The online version of this article (doi:10.1007/s10811-014-0342-2) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction Major hurdles to realising the potential of algal biomass as a source of sustainable feed for food production, biotechnology, and ‘green’ biofuel include having the ability to produce and extract the required biochemicals cheaply, efficiently and on an industrial scale (Greenwell et al. 2010 ). Of these, optimization of biomass production is widely held as a, if not the, critical step in the exploitation of high value products (Stephens et al. 2010 ; Borowitzka 2013 ). In contrast, the emphasis in studies of algal biotechnology has tended to be on increasing the relative content of the target biochemical. In reality, it is the optimisation of the production rate of those specific biochemical components per unit of effort, set against their market value, which is the critical issue. Rather less attention appears to have been given in publications to manipulating cell composition primarily for the purpose of optimising production of biofuels or as feed in intensive aquaculture, either via modification of growth conditions and/or of the genetics of the organisms themselves through GM. While the use of GM, transgenics and similar approaches have clear potential, concerns may be raised over various environmental risks (Flynn et al. 2010a , 2013 ). A more cautious approach is to make the most of wild strains, modifying growth conditions in order to maximise production of the required components. The other dichotomy in approaches is the use of natural versus artificial illumination. For high quality, high biosecurity and high repeatability of production, artificially lit enclosed bioreactors offer clear advantages. However, for truly intensive production, natural illumination is required else the financial if not the energetic costs of artificial lighting become prohibitive. Likewise, for local low-technology solutions, the use of solar illumination is most likely the favoured first option. In this paper, the term ‘biofuel’ refers to any form of excess carbon stored by the cell and surplus to its immediate requirements which can be extracted and exploited for use in fuel technology. Such energy-rich substances include fatty acids and other lipids for use in biodiesels plus carbohydrates for bioethanol (Fon Sing et al. 2013 ). It should be noted though, that (depending on the source organism) fatty acids are also potentially important dietary feedstocks, especially as polyunsaturated fatty acids (the conflict being that bioenergy favours short chain saturated fatty acids). The contrary use of this algal biomass is for protein or high value compounds such as photo-protection pigments. While undoubtedly, a biorefinery approach (Greenwell et al. 2010 ) gives maximum flexibility, ultimately whether a particular batch of product is intended to be C- or protein-rich presents a basic division point in the process. Finding the ideal combination of light and nutrient availability to encourage production of excess carbon versus protein-rich biomass is not a trivial exercise; optimising for biofuel production does not follow directly as a result of optimising biomass production. This is because maximum growth and enhanced lipid production tend to be mutually exclusive (Flynn et al. 2010a ; Scott et al. 2010 ). Lipids accumulate when algae are N stressed (Flynn et al. 1993 ), a condition that is contrary for rapid biomass growth but one that can be exploited when developing strategies to maximise yields of oils and fatty acids by manipulating cell physiology (Li et al. 2008 ; Beer et al. 2009 ; Rodolfi et al. 2009 ; Greenwell et al. 2010 ). Optimal conditions may be achieved through selection of dilution rates in continuous culture systems to balance growth and N limitation, or in batch culture systems through a two-stage process whereby biomass is allowed to grow optimally and is subsequently N starved. However, there is the added complication that a dense N-starved culture is typically too self-shaded to allow sufficient light penetration to maximise the lipid yield within a reasonable time frame, if at all (Flynn et al. 2010a ; Su et al. 2011 ). The fundamental requirement for high levels of irradiance to enable rapid biomass and then, as applicable, excess C production, is of particular concern for growth using natural illumination at higher latitudes. Lower solar elevation and shorter winter days, combined with increased average cloud cover, decrease the amount of light available compared to locations at equatorial and intermediate latitudes. Requirements for artificial light to augment natural illumination need to be kept to a minimum to decrease costs. Thus, if one is to attempt cultivating algae on a commercial scale in, for example, northwest Europe, using methods found effective in the Southern USA or Australia, the first step should be to adjust expectation of yields to match local light availability, and modify operations accordingly. To date, estimates of production at a given location have typically been made by calculating (with varying degrees of sophistication) photosynthetic activity based on local irradiance profiles and presumed algal photo-efficiency. However, the resulting broad range of projections has produced much uncertainty as to what is realistically achievable (Williams and Laurens 2010 ; Ritchie and Larkum 2012 ). What such calculations lack is the ability to adequately capture the physiological response of cells to the interplay between external environmental factors; for instance, to the shifting balance between light and N limitation, described above, which has so much bearing on lipid production rates. A more effective way of simulating these dynamics is by employing mechanistic acclimative models of algal growth. As an aid to developing improved cultivation methods, mathematical models of algal growth can be utilised to build and run virtual systems simulated in silico , providing theoretical projections of possible yields. Such computer-based experiments can be performed in only a fraction of the time and cost of the ‘real world’ equivalent. The model on which this investigation is based (Flynn 2001 ) is an acclimative mechanistic model that is able to capture the dynamics of multi-nutrient interactions and can be modified to function within a wide range of virtual environments. While models exist for simulating detailed aspects of algal physiology (Novoderezhkin and van Grondelle 2010 ; Gorbunov et al. 2011 ; Papadakis et al. 2012 ; Xin et al. 2013 ) the critical issue here is relating biomass production to nutrients and light. This requires an intermediate level of description, between the extreme simplicity of models typically used in earlier analyses (Weyer et al. 2010 ) and systems biology approaches that cannot yet capture growth dynamics (not least because we lack the data sets to test such models). Features of the model used here include potential for a full representation of variable elemental stoichiometry (C, N, P, Fe and, for diatoms, Si) driven by variable temperature, nutrient (NH 4 \n + , NO 3 \n − , PO 4 \n − , bioavailable Fe, and SiO 4 ) and light regimes (describing variable Chl:C with photoacclimation and nutrient status). Its effectiveness has been demonstrated repeatedly against data for various species, for diverse aspects of growth under varying conditions including (but not limited to) biomass and photoacclimation (Flynn et al. 2001 ; John and Flynn 2002 ), nutrient quotas and transport controls (Flynn 2008 ), production of dissolve organic matter (Flynn et al. 2008 ) and applications to ecology (Fasham et al. 2006 ; Flynn 2010 ). The production of excess C (carbohydrate and fatty acids) can also be simulated (Flynn et al. 2013 ). For the first time, this model is applied to an investigation into the effects of multi-nutrient interactions on potential algae production using solar irradiance over a broad range of latitudes over the Earth’s surface. The projected geographical and seasonal variations in both biomass and biofuel (excess C) production are explored under a range of operational conditions, providing a framework for future more detailed and empirical studies, as well as sensitivity and financial analyses. What we present is thus a best case scenario, assuming no downtime for maintenance or system failures and also that certain engineering challenges are met.",
"discussion": "Discussion Comparing the model projections with empirical evidence This work seeks to provide a more quantitative assessment of the scope for solar-powered algal biomass production than has hitherto been published. Absolute peak of year-averaged C-biomass productivity in these simulations was 2.4 gC m −2 day −1 . More typically, mean AP (Fig. 3 ) fell between 0.8 and 2.3 gC m −2 day −1 , depending on strain configuration and geographic location. Taking the C/dry weight biomass ratio to be 31 % (Heymans 2001 ) and assuming uninterrupted production could be maintained, this allows a rough estimate of a limit on annual areal production at slightly under 30 t dw ha −1 year −1 for a strain which undergoes one doubling during a 12:12 h light/dark cycle. For strains with faster maximum growth rates (along with a suitable choice of dilution rate), the absolute value of production should rise further. This projected peak value of 30 t dw ha −1 year −1 from our simulations falls within the mid-range of those reported for different real systems. It is in good agreement with the results of Jimanez et al. ( 2003 ) who report annual production of 30 t dw ha −1 year −1 in raceways in Southern Spain. Olguin et al. ( 2003 ) report an average production of 11.8 g m −2 day −1 in Mexico over the course of a year cultivating Spirulina using animal waste, which would equate to around 40 t dw ha −1 year −1 of biomass if production could remain uninterrupted over the whole year. Productivity of 60 t dw ha −1 year −1 of Pleurochrysis carterae in flat ponds has been reported (Moheimani and Borowitzka 2006 ), and cited as an example of maximal productivity (Williams and Laurens 2010 ), but this weight includes 10 % of calcium carbonate in the form of coccoliths and remains very much the exception rather than the rule. At the other end of the scale, García-González et al. ( 2003 ) achieved production of Dunaliella equating to around 6 t dw ha −1 year −1 . This compares favourably with the projected AP for the slowest growth rate optimised for in Fig. 1 (0.65 gC m −2 day −1 for U \n m = 0.346, comparable to that of Dunaliella ) which is approximately 7.5 t dw ha −1 year −1 . For other system types, there are reports of AP up to 60 t dw ha −1 year −1 for tubular PBRs (Fernández et al. 1998 ; de Schamphelaire and Verstraete 2009 ; Rodolfi et al. 2009 ) and nearly 40 t dw ha −1 year −1 for hybrid systems (Huntley and Redalje 2007 ), dependent upon the strain cultivated. The more typical model predictions for AP above of 3–8.4 tC ha −1 year −1 (around 10–27 t dw ha −1 year −1 using the above estimation) are of the same order as estimated by Ritchie and Larkum ( 2012 ) who measure net photosynthesis for three algae species from measurements of light attenuation in cultures of varying optical depths. In conclusion, this all instills confidence that the model used in this study is producing plausible projections. Potential for biofuels production Our simulations indicate a maximum potential biofuels production rate of 0.9 g Cex C m −2 day −1 , attainable at latitude 15° with a system of optical depth of 0.1 m and a dilution rate of D = 0.25 day −1 , under an f/4 nutrient regime (see Fig. 6b ). This assumes that all Cex C is of use for biofuels production. The maximum Cex C content of the simulated microalgae was 63 % of algal C-biomass (compare Fig. 6 with Fig. S3 ) but this coincided neither with peak AP (with 9 % Cex C ) or peak AXP (48 % Cex C ). The typical Cex C content using f/4 medium ranged between 10 and 60 % for strain S and 30 and 60 % for strain F, depending on latitude and dilution rate. For comparison with strain S, lipid content of up to 60 % has been measured in strains of Nannochloropsis under N deprivation (Rodolfi et al. 2009 ) whereas for Scenedesmus (cf. strain F) optimised lipid content has been reported at 58 % (Mandal and Mallick 2009 ). A review of reported lipid content values is provided by Mata et al. ( 2010 ). The shallow nature of the optimal depth required to assure a production of biofuels becomes a challenge if considering flat raceways for cultivation; requirements for adequate mixing and high susceptibility to evaporation and temperature fluctuations in such shallow ponds mean that it is often impractical to operate raceway systems with depths less than 0.15 m (Tredici 2007 ; Ritchie and Larkum 2012 ). Whilst not detrimental to AP per se, the subsequent lower VP resulting from this pragmatic limitation decreases the potential profitability by increasing demand for water and nutrients and increasing harvesting costs. Furthermore, the results in Fig. 7a imply there is also a direct adverse effect on AXP. Increasing the optical depth to 0.15 m (while keeping D fixed) leads to a decrease in AXP of between 10 and 25 % (depending on latitude) compared to the potential peak value. Increasing depth further to 0.2 m results in a halving (or worse) of AXP compared to production under optimal conditions. To some extent, this can be mitigated by adjusting the dilution rate appropriately, as Fig. 8b shows; slowing the dilution rate from D = 0.25 to 0.1 day −1 limits the decrease in AXP from peak values to about 20 %. Even so, if the system which produced the peak value in AXP quoted above was limited in practice to a depth of 0.2 m with dilution slowed to D = 0.15 day −1 , peak AXP would not exceed 0.8 g Cex C m −2 day −1 . These factors, and given that the model is producing results consistent with data from real systems (“ Comparing the model projections with empirical evidence ” section), appears to provide a robust estimation of the upper potential for solar-powered microalgal biofuels production of 3 t biofuels ha −1 year −1 , which equates to ~4,000 L ha −1 year −1 assuming a carbon fraction of 720 gC L −1 (which is typical for diesel fuels (Miguel et al. 1998 )) and that all of the excess C can be recovered and is in the form of lipids. While outperforming many land-based crops, these results imply algae are not appreciably more productive for biofuels and can be even less so in comparison with, as an example, palm oil (Chisti 2007 ; Schenk et al. 2008 ; Mata et al. 2010 ; Scott et al. 2010 ). This upper limit is in agreement with the calculation performed by Walker ( 2009 ) and far below many estimates of theoretical limits (Weyer et al. 2010 ). In reality, it is unlikely (if not impossible) that optimal culturing conditions can be maintained long enough to achieve the kind of results for biomass and biofuels production obtained in these simulations. To be able to quantify this further requires a detailed sensitivity analysis of risk factors. Even so, should it be possible to overcome the technical difficulties, the physiological limits on cell growth constrain the potential for algae as a feedstock for biofuels. As a result, the potential for biofuels production from microalgae appears of questionable commercial viability, unless a step change can be attained in algal physiology through GM, with all of its attendant risks. For instance, Flynn et al. ( 2013 ) demonstrated through simulation how engineering strain characteristics to allow greater capacity for photosynthetic efficiency coupled with a decrease in the maximum Chl:C ratio could boost productivity by up to five times that of natural strains. They projected a maximum Cex C production rate of AXP = 7.5 g Cex C m −2 day −1 = 20,000 L ha −1 year −1 of biodiesel. At the same time, they also demonstrated how the creation of such unpalatable, highly productive strains (desirable traits for biofuels production) could easily lead to harmful, even catastrophic, blooms if they escaped into nature. To date, even with more optimistic production estimates, there remains much uncertainty for the economic potential for microalgal biofuels production (Liu et al. 2012 ; Sills et al. 2013 ). In life cycle analyses, this uncertainty is dominated by sensitivity to the algae’s lipid content and growth rate (Stephenson et al. 2010 ; Davis et al. 2011 ). Unfortunately, the biological modelling components within otherwise complicated LCA scenarios are invariably based on assumptions and generalisations derived from literature which lead to projections of several hundreds of tons of biomass produced per hectare per year (Williams and Laurens 2010 ) whereas in practice (as seen from the references above) 60 t dw ha −1 year −1 is the highest claimed to date. Even if 60 t could become the rule rather than the exception, only a fraction of that (ca. at most 50 %) can be expected to constitute stock for biofuels. Our results indicate production of biomass AP below 30 t dw ha −1 year −1 and biofuels feedstock AXP up to 8 t dw ha −1 year −1 . In consequence of all of these interacting events, conducting a full LCA on the commercial viability of the whole process (whether for biomass, biofuels or other products) requires an integrated approach taking into account the physiology of the microalgae that lay at the heart of the whole endeavour. At present, LCAs take scant regard of this issue and in consequence may be at significant variance from reality. It is likely that the inherent uncertainty will remain unresolved until LCAs become coupled to mechanistic models (such as the one used here) that can more adequately capture the dynamic physiological subtleties of microalgal growth. Combining these informative but differing computational approaches can provide a powerful tool that will allow operators to explore realistic options leading towards improved production. Areal vs volumetric production The emphasis above has been upon areal production of biomass (AP) and of excess C as biofuels (AXP). In the Appendix are the corresponding volumetric production values (VP, VXP, respectively). For commercial operations, it is important to maintain an optimum balance between AP and VP. However, this ideal is conflicting as the highest VP requires very low optical depths, which do not then permit high AP (see Flynn et al. 2010a ). In oceans, with optical depths of many tens of meters, VP is extremely low, but AP by fast-growing phytoplankton at upwelling zones (ca. 3–4 gC m −2 day −1 (Field et al. 1998 )) can match rates in shallow ponds (Flynn et al. 2013 ) in short bursts during spring blooms. Figure S1 illustrates how a high VP requires a shallow optical depth to prevent light limitation and this has the added benefit of diminishing the demands for nutrients and water, and hence harvesting costs. The trade-off comes as the resulting low volume minimises AP. Increasing depth to boost AP suppresses VP but the rate of depth increase initially outpaces the rate of VP decrease and so the AP continues to rise. After a certain point, VP decreases in proportion to the increase in depth which leads to the saturation of AP seen in Fig. 4 . For Cex C , the corresponding rise in AXP (Fig. 7 ) as VXP falls (Fig. S4a ) initially follows the trend for AP as optical depth increases. However, beyond a critical optical depth (~0.1 m) light rather than nutrient becomes the limiting factor; Cex C production is suppressed and VXP decreases faster than the depth increases leading to the fall in AXP. Production of C-rich products (e.g. biofuels) is, therefore, more sensitive to the conflict between areal and volumetric production than is biomass production. This conflict will extend directly to costs for space (and/or PBR infrastructure) and in preparing and handling different volumes of water/algal suspension and nutrient loadings. Flynn et al. ( 2010a ) described this interaction using a function relating AP and VP to what they termed a commercial production index. While this provided a single index, in the commercial world the costs of land, energy, nutrients, and other resources would apply differential weights to AP vs VP. The results from the simulations presented here enable a number of useful conclusions to be drawn in this regard. Most notably, the routes to maximising production of biomass are not the same as those for maximising fatty acid/biofuels production. That said, while individual needs may vary, the optimal depth for commercial cultivation of wild-strain (non-GM) phototrophic microalgae in a facility intended for multiple applications should be approximately 0.1 m (a value consistent with that suggested by García-González et al. ( 2003 ) and Ritchie and Larkum ( 2012 ) who also place an upper limit on useable pond depth around 25 cm) coupled with the use of nutrient loads of around f/2 containing 12.35 mg N L −1 and 1.11 mg P L −1 (Guillard and Ryther 1962 ) for biomass production, and f/4 for biofuels production. Further, in general, stimulating biofuels production requires the combination of a fast-growing strain, and nutrient deficiency, which is promoted by shallow optical depth and relatively slow dilution rates. Latitudinal impacts Not surprisingly, production at high latitudes is projected to be far more seasonally dependent than at lower latitudes, as seen for both biomass in Fig. 2 and Cex C in Fig. 5 . However, while biomass production in mid-winter may be so poor as to likely not be commercially viable, the longer summer days have potential to provide a window for increased production over the summer months sufficient to ensure viability. That may be especially so if the intended use of the biomass in support of seasonal aquaculture activities. This paints a qualitatively similar picture to the gross photosynthesis calculations of Ritchie ( 2010 ) and to Williams and Laurens ( 2010 ) who suggest a lack of sufficient irradiance over winter months restricts areal production at high latitudes to little more than half of that possible at equatorial latitudes, compared to our prediction of around 60 % of maximum. However, Williams and Laurens’ estimate of absolute values for production (obtained by assuming either a 3 or 10 % bioenergetic yield with a biomass calorific value of 24.7 kJ g −1 dw) are larger than the mechanistic model calculations by a factor of 10. Our values are more keeping with empirical values from the literature (‘ Comparing the model projections with empirical evidence ’ section). The optimal dilution rate depends primarily on the maximum growth achievable by the strain cultivated. Even though growth may be conducted at low dilution rates, a high U \n m is a desirable trait to select or engineer into microalgal strains (Flynn et al. 2013 ). However, this trait is likely to be selected against during long-term enforced slow growth in continuous culture systems (Flynn 2009 ). Away from tropical latitudes, location becomes an additional factor. The extent to which it does so also depends upon the maximum growth rate; production using slower growing strains is more sensitive to the choice of dilution rate with increasing latitude than it does for a faster growing strain; the optimal dilution rate progressively decreases the further from the equator the facility is situated (Figs. 3 and 6 ). Furthermore, as faster-growing strains are less sensitive to the choice of dilution rate, the most appropriate dilution rate may not necessarily be the one that supports maximum biomass yield. Figure 1a shows that decreasing the dilution rate for the strain with U \n m = 1.386 day −1 from D = 0.35 to 0.2 day −1 uses <60 % of the nutrients and water but still returns 90 % of peak AP (1.8 cf. 2 gC m −2 day −1 ). Such a saving of resources is likely to impact significantly on commercial viability, especially as nutrient prices increase. System optimisation The results from Fig. 1 , and indeed the seasonal variability in production at high latitudes, imply a single objective optimisation method may be insufficient. A more sophisticated and effective method could be to use multi-objective optimisation to balance between maximising biomass production and minimising resource consumption, and also consider financial inputs and outputs. At extremes, one could consider changing algal strains or system optical depths (Olguin et al. 2003 ; Moheimani and Borowitzka 2007 ), but more readily changed are facility operational procedures such as dilution rate. While higher-plant crops are grown in what amounts to discontinuous culture, industrial-scale microbial growth is most commonly within continuous culture systems. All of the simulations performed here have been run in such a chemostat mode, with continuous dilution and harvesting at a constant rate. While there are problems with using such an approach (notably strain selection to match maximum growth rate to dilution rate, and the risk of establishing pest-predators), there are also distinct logistic problems in discontinuous approaches. These include the necessity to rapidly drain and harvest large volumes of medium containing the biomass, and replace the same volume with fresh medium and nutrients. Exposure of the newly diluted remaining culture to high light then risks photodamage, especially if the organisms are nutrient stressed (Geider et al. 1993 ). Improvements to the use of continuous dilution methods as simulated here would be to consider seasonal changes to the dilution rate and/or through the introduction of discontinuous harvesting methods into the simulations. From our results, it appears that an optimisation of dilution rates separately for winter and summer months could be sufficient. For instance, from the results in Fig. 2b \n i and iv , it seems more appropriate to run with a substantially slower dilution rate in winter at high latitudes and increase dilution as production ramps up later in the year. To fully automate the optimisation process, a real-time regulation of production rates is needed. Computationally, this can be achieved using a simple predictor-corrector. At regular intervals, a prediction is made of the output at the end of a time period t + m∆ t based on the current dilution rate and compared to the output for a set of dummy rates. Whichever gives the best result becomes the rate for that time period. Methods of intelligent harvesting (whether manual or automated) would have to rely on real-time monitoring of culture conditions, with several measurements taken simultaneously. Monitoring and regulation of external nutrient levels would be quite straightforward, deploying nutrient probes directly exposed to the culture medium. Regulating dilution based on algal physiology is more problematic, though monitoring of biomass and photophysiology using F \n v / F \n m to monitor the efficiency of the PSII photosystem are obvious starting points. Measurement of the fluorescence emission spectrum can reveal levels of nutrient stress in the culture (Masojídek et al. 2000 ); a threshold value could be used as a trigger for further nutrient injection. Light absorption is an indicator of total biomass (Griffiths et al. 2011 ) and measurement of changes in turbidity can provide an estimation of growth rates, while analysis of the absorption spectrum can quantify the amount of chlorophyll per unit of biomass. Thus, if a measurement of turbidity and/or F \n v / F \n m indicates an aberrant change in production then the dilution rate can be automatically adjusted to compensate. Intelligent control of dilution rates and nutrient addition is one way to optimise yields, but a more radical control scenario would enable switching between periods of continuous and discontinuous operation. This leads to further questions as to what the optimal dilution and harvesting strategies may involve in different modes, and how these may relate to other factors such as the control of pests. These topics will be considered in future papers."
} | 7,542 |
38434856 | PMC10905720 | pmc | 6,209 | {
"abstract": "Bioplastics are one\nof the answers that can point society toward\na sustainable future. Under this premise, the synthesis of polymers\nwith competitive properties using low-cost starting materials is a\nhighly desired factor in the industry. Also, tackling environmental\nissues such as nonbiodegradable waste generation, high carbon footprint,\nand consumption of nonrenewable resources are some of the current\nconcerns worldwide. The scientific community has been placing efforts\ninto the biosynthesis of polymers using bacteria and other microbes.\nThese microorganisms can be convenient reactors to consume food and\nagricultural wastes and convert them into biopolymers with inherently\nattractive properties such as biodegradability, biocompatibility,\nand appreciable mechanical and chemical properties. Such biopolymers\ncan be applied to several fields such as packing, cosmetics, pharmaceutical,\nmedical, biomedical, and agricultural. Thus, intending to elucidate\nthe science of microbes to produce polymers, this review starts with\na brief introduction to bioplastics by describing their importance\nand the methods for their production. The second section dives into\nthe importance of bacteria regarding the biochemical routes for the\nsynthesis of polymers along with their advantages and disadvantages.\nThe third section covers some of the main parameters that influence\nbiopolymers’ production. Some of the main applications of biopolymers\nalong with a comparison between the polymers obtained from microorganisms\nand the petrochemical-based ones are presented. Finally, some discussion\nabout the future aspects and main challenges in this field is provided\nto elucidate the main issues that should be tackled for the wide application\nof microorganisms for the preparation of bioplastics.",
"conclusion": "6 Conclusion Throughout\nthis review, it could be observed that bioplastics can\noffer a convenient way to utilize food and agricultural waste as sources\nto biosynthesize polymers using microbes. Many microorganisms can\nbe used for the synthesis of polymers or monomers to produce PLA,\nPHAs, PLGA, and PBS, among many others. These factors unlocked a broad\nrange of possibilities for the synthesis of polymers that can serve\nas an alternative to petrochemical-based materials. Hence, the investment\nin such technology is an important step toward a more sustainable\nfuture as the manufacture of these bioplastics can simultaneously\nhelp with waste management as well as decrease the strain on the demand\napplied to petrochemical-based polymers. Bioplastics can be employed\nin many sectors such as packing, cosmetics, pharmaceutical, biomedical,\nmedical, sanitary, and agricultural. On top of that, biopolymers such\nas PLA, PGA, chitin, and cellulose for instance are biodegradable\nwhich can promote a circular economy with a considerably reduced generation\nof waste and carbon footprint. It has been proposed that biopolymers\nshould play a major role in the future as they have already been implemented\nin the market such as in the case of PLA and PHAs, for instance. Yet,\nthese materials are still in their infant phase and further investment\nis still required to diminish their overall production cost as well\nas make their properties more competitive against petrochemical-based\npolymers. Even though this process is inherently challenging, as it\nrequires optimization in every step of the production, several parameters\ncan be varied such as type of microbe strain, substrate, polymerization\ntechniques, compositing, processing, and manufacturing which can lead\nto polymers with a wide range of properties. Yet, these steps also\nhave their challenges that may hinder the optimal manufacturing of\nbiopolymers when compared to polymers obtained without the need for\nmicroorganisms. First, there is a need to improve the yield and productivity\nas it can be affected by the substrate, environment parameters, and\nthe presence of different strains in the microbial system. Second,\nenhancing the polymer synthesis through genetic engineering can be\na time-consuming process. Third, finding the optimal balance between\na suitable feedstock for the bacteria as well as utilizing waste biomass\nto promote a more economically viable and sustainable process. Fourth,\nthe processing and purification processes to extract the polymer from\nthe bacteria can be energy-demanding and costly. Hence, optimizing\nthese processes is also required to increase their competitivity. 240 Yet, despite these challenges the production\nof biopolymers such as PLA, PBS, PBAT, PHA, and starch-blend is expected\nto increase up to 2025. 241 − 243 However, even though the production\nof biopolymers is leaning upward is still challenging to confirm if Thus, further research on bioplastics to make their properties\nmore competitive against petrochemical-based polymers along with the\ndecrease in cost can lead to a considerable shift in the market as\nthey can potentially solve issues related to the waste generation\nand aggressive consumption of nonrenewable resources.",
"introduction": "1 Introduction Over the years there has\nbeen a continuous increase in the consumption\nof plastics mostly derived from nonrenewable sources as its production\nreached around 338 million tons in 2019 which was almost 6.5 times\nhigher than that from 1975. 1 Even though\nthere has been an effort to recycle plastics, in the United States,\nit only accounts for 10% of the amount wasted. 2 Such conditions lead to a considerable strain on the manufacturing\nand nonrecycling of plastics resulting in ending up in ecosystems\nand landfills. 3 Some of the major issues\nwith these plastics are their long degradation time (over a century\nfor most of the plastics) and the formation of harmful macro and microplastics\nduring decomposition which discharge into the ecosystems. Continuing\nwith a linear economy for plastic production can culminate in the\nexhaustion of nonrenewable resources along with environmental and\neconomic issues. There has been an effort to increase the production\nof biobased and biodegradable materials which is expected to grow\nin the next few years ( Figure 1 ). 4 Based on that, it is worth\ndiscussing the difference between biopolymers and bioplastics. In\nthis sense, biopolymers can be considered as a broader range of materials\nthat originate from natural sources or can be synthesized by living\norganisms, hence it includes carbohydrates (i.e., cellulose, chitin),\nproteins (i.e., silk, collagen), nucleic acids (i.e., DNAs and RNAs),\namong others. These materials can be found in living organisms or\nobtained from biomass. The term Bioplastic can be defined as a more\nspecific class of biopolymers that are plastics obtained from renewable\nresources or through the biosynthesis of microorganisms. Several renewable\ncarbon sources can be used for the synthesis of bioplastics such as\nstarch from corn, potatoes, cassava, sugar cane, cellulose, vegetable\noils, algae, and agricultural biowaste. In this sense, some of the\nmost known examples of bioplastics obtained from bacterial metabolic\nprocesses are polyhydroxyalkanoates (PHAs) which is a group of polymers\nthat includes polyhydroxybutyrate (PHB), 5 polyhydroxyvalerate (PHV), 6 polyhydroxyhexanoate\n(PHH), 7 polyhydroxyoctanoate (PHO), 8 poly(3-hydroxybutyrate- co -3-hydroxyhexanoate)\n(PHBH), 9 among others. The previous examples\nof bioplastics are biodegradable; however, there are also nonbiodegradable\nbioplastics, such as bio polyethylene (PE), bio polypropylene (PP),\nand bio polyethylene terephthalate (PET), which can be obtained either\nfrom renewable or petrochemical sources. Figure 1 Worldwide production\nof bioplastics from 2021 to 2022 along with\nthe forecast up to 2027. Adapted with permission. 4 Copyright 2022, European Bioplastics. Under this premise, one feasible option to work around this situation\nis based on the preparation of biodegradable polymers derived from\nrenewable sources or biowaste as it can ease the burden on their petrochemical-based\ncounterparts while promoting a circular and sustainable economy. That\nprocess can be performed through the use of bacteria which are capable\nof synthesizing a plethora of polymers such as polysaccharides, polyesters,\npolyamides, and even polyphosphates. 10 Bacteria\ncan produce polymers mainly in two ways. First, it can break down\nlarger molecules of biowaste into a monomer that can be further used\nfor polymerization. Second, bacteria can be inserted into a type of\nenvironment that promotes the synthesis of a polymer that can be extracted.\nUsually, both processes take place through fermentation. Some examples\nof the first case, in which bacteria perform a fermentation process\nto obtain a monomer that can be later chemically polymerized are poly(lactic\nacid) (PLA), some starch-based polymers (TPS), and polybutylene succinate\n(PBS) which consist, in its majority, of biodegradable aliphatic thermoplastic\npolyesters. It is worth noting that, even though the PLA is mostly\nproduced through a chemical polymerization process based on the ring-opening\nreaction of lactide, there have been some recent approaches that proposed\na one-step fermentation process for the biosynthesis of PLA through\nmetabolically engineered microorganisms. 11 For that, two main enzymes must be present which are the propionyl-CoA\ntransferase and PHA synthase. The process consists of the conversion\nof lactic acid into lactyl-CoA through the enzymatic activity of propionyl-CoA\ntransferase. Then, lactyl-CoA is polymerized by the PHA synthase.\nSome of the polymers that can be synthesized by bacteria are dextran,\nxanthan, and polyhydroxyalkanoates (PHAs), which include a vast number\nof polymers, such as polyhydroxybutyrate (PHB), polyhydroxyvalerate\n(PHV), polyhydroxyalkanoate (PHO), among others. PLA is commonly\nobtained from the fermentation of sugars or starch\nderived from carbohydrates from plants. Several sources which include\ncorn starch, sugar cane, wheat, and rice straws can be used as starting\nmaterials to produce PLA. Such components are based on polysaccharides\nwhich contain cellulose and hemicellulose that can be depolymerized\ninto sugars either through chemical or enzymatic routes. The obtained\nsugars can be fermented into lactic acid to be polymerized into PLA.\nAnother attractive biodegradable polymer is PBS which is also a thermoplastic\npolyester that can be manufactured from food waste. 12 It can also be obtained from the same sources as PLA, as\nwell as feedstock from algal, plant, or vegetable oils. It is most\nsynthesized from the polycondensation reaction between succinic acid\nand 1,4-butanediol. It is worth noting that, even though 1,4-butanediol\ncan be derived from petrochemicals it can also be obtained from the\nfermentation of sugars and molasses from beet, sugar cane, corn starch,\ncorn stover, and wheat straw. The PHAs are another widely studied\nclass of biodegradable polyesters that are produced by bacteria usually\nwhen there is a nutrient limitation related to the lack of P, N, and\nO. This polymer displays great versatility in terms of the substrate\nand bacteria that can be used for its synthesis. In this sense, some\nof the substrates are agricultural waste, fatty acids, 13 olive oil pomace, fermented molasses, 14 and paper and palm mill waste. 15 , 16 There is a plethora of microorganisms that can synthesize PHAs from\nthe mentioned substrates which include Pseudomonas sp. , 17 Rhodobacter sphaeroides , 18 Rhizobium sp ., 19 Ralstonia eutropha , 20 Enterobacter sp ., 21 among many others. Such versatility in substrate\nand microorganisms available has led to well-established manufacturing\nprocesses biodegradable and biobased polymers such as PLA, PHAs, PBS,\nand starch blends. In addition, there are also some biodegradable\npolymers derived from nonrenewable sources such as polyethylene glycol\n(PEG), polycaprolactone (PCL), poly(trimethylene carbonate) (PTMC),\nand polybutylene adipate coterephthalate (PBAT) which have also gained\nsome space in the market. These biodegradable polymers present several\nattractive properties such as a broad range of mechanical properties,\nprocessability through different techniques, biocompatibility, and\nstructural versatility that allow their introduction into the market.\nSuch features led to the production of around 2.22 million tons of\nbiodegradable polymers worldwide in 2022 which are composed of several\nindustries including food packing, cell scaffolds for tissue engineering,\ndrug delivery systems, face masks, cosmetics, agricultural products,\nand many others ( Figure 2 ). 4 , 22 Figure 2 Biodegradable polymers and their main applications.\nAdapted with\npermission. 22 Copyright 2022, Springer\nNature."
} | 3,174 |
34901665 | PMC8655920 | pmc | 6,211 | {
"abstract": "Individual bacteria communicate by the release and interpretation of small molecules,\na phenomenon known as quorum sensing (QS). We hypothesized that QS\ncompounds extruded by Photorhabdus could be interpreted\nby Bacillus —a form of interspecies communication.\nWe interrogate the structure–activity relationship within the\nrecently discovered pyrone QS network and reveal the exquisite structural\nfeatures required for targeted phenotypic behavior. The interruption\nof QS is an exciting, nonbiocidal approach to tackling infection,\nand understanding its nuances can only be achieved by studies such\nas this.",
"conclusion": "Conclusions In conclusion, some 25 natural 41 and\nunnatural pyrones were synthesized. 42 − 44 The naturally (in P. luminescens ) occurring PPYA compound 1 was shown to enhance biofilm formation in B. atropheaus . In direct contrast, replacement of\nthe isobutyl group at the C6 position with a simple methyl group gave\ncompounds that induced antibiofilm activity. Indeed, antibiofilm activity\nwas also noted for all other compounds, even those with an isobutyl\ngroup at C6 and differing from 1 by ±CH 2 in the alkyl chain. Swarming inhibition activity appeared to require\nless structural specificity compared to the biofilm activity, although\na requirement for specific chain length at C3 did emerge as critical\nfor inhibition. The mechanistic basis of the behavioral changes\nidentified in response\nto the pyrone signals and their derivatives remains to be elucidated.\nA LuxR-type receptor–ligand interaction would be complex and\nlikely multifaceted, considering the varied impacts of the PPY derivatives\nsynthesized when compared to the native PPYA signal. QS plays a role in biofilm development and swarming motility in B. subtilis , 33 , 45 , 46 with AI-2 signaling recently reported eliciting a biofilm-dependent\nresponse through the LuxS receptor under specific environmental conditions. 47 A LuxR-type receptor–ligand interaction,\nsuch as that described for the pyrones in Photorhabdus , would equally be complex and likely multifaceted. The activity\nand specificity of the natural pyrone signal 1 suggest\nthat pyrones may have an interspecies communication role similar to\nthat seen for the alkyl hydroxyquinolone signal molecules, HHQ and\nPQS, in P. aeruginosa , which also display\nsimilar specific structure–activity relationships. 20 , 21 , 26 It is also worth noting that\nboth HHQ and PQS modulate the behavior of a broad range of co-colonizing\npathogens, yet the MvfR/PqsR receptor for these two QS molecules has\nnot yet been identified outside of P. aeruginosa . The pyrone signal and derivatives presented in this report showed\ninterspecies activity in the low micromolar range, eliciting a dose-dependent\nphenotypic response at 50 μM, in contrast to the nM range of\nendogenous signal activity observed in P. luminescens . 14 This is consistent with the activity\nrange of the HHQ and PQS signals, which are active in the nM range\nagainst their native receptor (PqsR) in P. aeruginosa , 23 yet require concentrations in the\n10–100 μM range to elicit an interspecies response. 20 , 21 , 26 An important consideration here\nis the degree to which these compounds are naturally soluble in assays\nor indeed within the particular microbial community or ecosystem within\nwhich they are found. As an analogy, the solubility of both HHQ and\nPQS is significantly enhanced by endogenous biosurfactants called\nrhamnolipids, which are produced by P. aeruginosa . 48 It is unclear as yet whether a similar\nphenomenon underpins the biological activity of photopyrones at the\ninterspecies level as reported here, or indeed their interaction with\nsolo LuxR receptor proteins. Deciphering the breadth of signal-mediated\ninteractions that underpins\nmicrobial communication is an important endeavor as we attempt to\nunderstand the “community networks” that sustain microbiomes\nin health and disease. 49 The pyrone derivative\ndescribed here may have applications against other pathogenic bacteria,\nwhich possess a LuxR solo protein, and this will be the focus of further\nstudies. When this is the case, an extensive investigation of pyrone\nproduction in microbial communities would be warranted, particularly\nin light of the network of orphan LuxR proteins that exist in pathogenic\norganisms. The key finding of this study, the capacity for pyrones\nto elicit behavioral changes in a Gram-positive organism at a low\nmicromolar concentration, points to the role of this new class of\nmolecular signal in the interspecies interactome. Elucidating that\nrole, and the extent to which it governs virulence and pathogenesis\nin competing organisms will require an interdisciplinary approach.",
"introduction": "Introduction Bacteria can coordinate\ntheir collective behavior through an elaborate\ncommunication network. 1 This remarkable\nrealization has offered new insights into the complexity of microbial\ninfections. 1 Cooperation of/within microbial\nconsortia is governed by the extrusion and perception of small-molecule\nsignals (called autoinducers—AIs), a phenomenon known as quorum\nsensing (QS). Thus, bacteria monitor their external environment and\ncan significantly alter gene expression to act as a single multicellular\norganism if required. 2 , 3 These interactions yield the capacity\nto accomplish tasks that are futile when performed by an individual\nbacterium 4 —typically bioluminescence,\nsecondary metabolite synthesis, and perhaps, most importantly, biofilm\nformation and virulence factor production. 5 − 8 Through this ability to coordinate\nbehavior and form biofilms, QS allows bacteria to become more potent\npathogens, less susceptible to antibiotic treatments, and often facilitates\nthe evolution of antibiotic resistance. 9 , 10 The\nprototypical QS model in Gram-negative bacteria is comprised\nof a LuxI-type AI synthase and a LuxR-type receptor, with N -acyl homoserine lactones (AHLs) being the most predominant\nclass of AIs generated and received by these proteins. 11 Although not fully understood, systems lacking\nany LuxI-like AHL synthase can still encompass proteins with homology\nto LuxR-type receptors, namely LuxR orphans or solos. 12 , 13 Seminal reports by Heermann and co-workers explicitly target the\nconcept that non-AHL producing bacteria can employ these receptors\nin the detection of other endogenously synthesized compounds and operate\na QS signaling pathway. 14 , 15 Critically, this group\nunearthed “photopyrones” (PPYs) participating in the\nQS network of the Gram-negative pathogen Photorhabdus\nluminescens through an orphan LuxR receptor. The quorum\nin this case activates a signaling cascade that transcribes the operon\nresponsible for the Photorhabdus clumping factor\n(Pcf)—a biofilm-like phenotype that plays a vital role in the\npathogenicity of these bacteria. 14 , 16 , 17 These findings encompass the first demonstration\nof 2-pyrones, specifically 3-alkyl-4-hydroxy-6-isobutyl-2 H -pyran-2-ones ( Figure 1 ), acting as signaling molecules in any bacterial strain. 14 Figure 1 Photopyrones (PPYs) A–H isolated from Photorhabdus\nluminescens. Although the findings specified\nthe detection of endogenously produced\nPPY signals, it is also known that orphan receptors can be used to\nrespond to exogenous signals, produced by coinhabiting microorganisms,\nfor example. 15 , 18 , 19 Following on from our work describing the interspecies and interkingdom\nactivity of the alkyl hydroxyquinolone signals 2-heptyl-4-quinolone\n(HHQ) and Pseudomonas quinolone signal (PQS) produced\nby Pseudomonas aeruginosa , 20 − 26 this report describes interspecies behavioral control exerted by\nthe P. luminescens pyrone signals on\na model organism, Bacillus atropheaus subtilis var. niger (globigii) termed B. atropheaus hereafter. Importantly, this Gram-positive, aerobic bacterium utilizes\nQS in the formation of biofilms, 27 making\nit a suitable model for analysis of signal-based interference with\ncell–cell communication. Entomopathogenic Photorhabdus and Bacillus species have demonstrated a close\ninter-relationship, working synergistically in some infections 28 , 29 and exchanging toxin systems in others. 30 The emerging interspecies and interkingdom role in cell–cell\ncommunication signals and the close relationship between Photorhabdus and Bacillus species led us to investigate the\npossibility that 2-pyrones, similar to those produced by the former,\ncould exert behavioral control over the latter. A bacterial\nbiofilm is a polymeric matrix structure that can grow\non living or inert surfaces. 31 Its formation\ninvolves a complex multistage process, underpinned by a signaling-based\ncommunication system that spans from the initial attachment phase\nthrough to maturation and dispersion. This lifestyle is adopted by\nup to 80% of infections in humans, 32 frequently\nallowing the bacterial colony to circumvent the host immune system\nand increase resistance to antibacterial agents. 10 Swarming is also governed by a complex QS-based communication\nsystem, which directs multiple aspects of behavior, including head\nto tail connections between individual bacterial cells in this and\nother Bacillus species. 33 Both of these essential phenotypes, which are linked to persistence,\nvirulence, and antibiotic resistance in bacteria, were investigated\nusing B. atropheaus . 34 , 35",
"discussion": "Results and Discussion The simplest of the natural signals,\nisolated from Photorhabdus , PPYA , ( 1 ) was initially synthesized and\ntested for biofilm and swarming motility altering activity. 36 , 37 Addition of PPYA 1 (50 μM) to media prior to\ninoculation with B. atropheaus cultures\nled to a dramatic increase in attached biofilm biomass when compared\nwith dimethyl sulfoxide (DMSO) and untreated controls ( Figure 2 a). We reasoned that a low\nmicromolar concentration would be in the physiological range and consistent\nwith previous studies investigating the interspecies role of other\nquorum sensing molecules. 20 , 38 , 39 In contrast, swarming motility was significantly repressed in the\npresence of PPYA , pyrone 1 ( Figure 2 b). This was independent of\nany growth-related effects, as determined by visual analysis of the\nbiofilm formed, indicating a shift toward stronger pellicle formation\nat the liquid–air interface (SI, Figure S1 ). The addition of a reduced concentration of compound 1 (10 μM) did not elicit a response from B. atropheaus at the same cell seeding density, indicating\ndose-dependency to the effects observed (SI, Figure S1 ). The influence of 1 on biofilm formation and\nswarming motility in B. atropheaus has\nextensive potential implications. This suggests that 1 has the propensity to induce interspecies activity beyond its inherent\nrole in the signal-producing Photorhabdus species. Figure 2 (a) Biofilm\nformation in B. atropheaus in the presence PPYA at 50 μM in DMSO presented\nas Abs 595nm following crystal violet staining. (b) Swarming\nmotility of B. atropheaus with PPYA or carrier control. Data presented are the average (±standard\nerror of the mean (SEM)) of three independent biological replicates.\nStatistical analysis was performed by one-way analysis of variance\n(ANOVA) with Bonferroni post hoc corrective testing (* p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.001). Following on from these\npromising results, we looked at preparing\na suite of analogues (known and novel) to probe the structure–activity\nrelationship. Diversification at C3 and C6 was targeted, along with\nsome modification of the pyrone core ( Scheme 1 ). 36 , 37 We then investigated\nthe impact of these analogues on biofilm and swarming properties in B. atropheaus . Scheme 1 (a–c) 2-Pyrone and 2-Pyridinone\nDerivatives Synthesized The Hantzsch ester used here\nis 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate. Method A: (i)\nhexamethyldisilazane (HMDS) (3 mL/mmol), N 2 , 80 °C,\n1 h and (ii) tetrahydrofuran (THF), n -BuLi (1.25\nequiv), alkyl bromide (2.3 equiv), −78 °C–rt, 16\nh. Method B: (i) N , N , N ′, N ′-tetramethylethylenediamine (TMEDA)\n(1.0 equiv), THF/hexamethylphosphoramide (HMPA) (5:1), n -BuLi (2.4 equiv), 0 °C, N 2 and (ii) alkyl iodide\n(1.8 equiv), 0 °C–rt, 16 h (see the SI for details). Based on the trend\ntoward enhanced biofilm formation observed with\nthe native pyrone signal PPYA 1 , we examined the impact\nof our 2-pyrone derivatives on the ability of B. atropheaus to form biofilms and attach to the surface of multiwell plates.\nWe started with analogues bearing a methyl group at C6 ( 2 – 17 ), which are relatively easy to synthesize.\nWhen tested at 50 μM, the following compounds exhibited antibiofilm\nactivity: 7 , 8 , and 9 —in\ndirect contrast to the activity of the natural pyrone signal 1 (SI, Figure S2 ). Although 6 led to a reduction in biofilm formation, this was not statistically\nsignificant when tested in 24-well plates ( Figure 3 a). It should be noted that all compounds\nexhibiting antibiofilm activity, contain a long alkyl chain at C3\n(C 7 –C 10 ). Subsequent validation analysis\nof lead derivatives through dose–response studies at 10, 30,\nand 50 μM confirmed the biofilm limiting activity of these compounds\n(SI, Figure S3 ). Figure 3 (a) Antibiofilm activity of pyrone derivative compounds at 50 μM in DMSO presented\nas Abs 595nm following crystal violet staining. Assays were\nperformed in 24-well plates. (b) Growth curve analysis of B. atropheaus in the presence of pyrone derivatives\n(see the SI for details). All data presented\nare the average (±SEM) of at least three independent biological\nreplicates. Statistical analysis was performed by one-way ANOVA with\nBonferroni multiple comparison post hoc corrective testing (* p ≤ 0.05; *** p ≤ 0.001). To determine whether the influence on biofilm was\nsimply a reflection\nof growth inhibition, B. atropheaus was grown in the presence of each compound and investigated temporally.\nIn terms of growth kinetics profiling, while compounds 8 and 9 had a growth-limiting effect on B. atropheaus , growth was not comparably affected\nin the presence of pyrones 6 and 7 ( Figure 3 b). This clearly\ndelineates the biofilm formation and growth, at least in compounds 6 and 7 . We, therefore, propose these compounds\nas lead compounds for antibiofilm activity, potentially working through\ninterference with signaling mechanisms. None of the other compounds,\nwith shorter or longer (linear, branched alkyl chains, aryl, heterocyclic)\ngroups at C3 showed considerable antibiofilm activity. For the activity\nof all 17 analogues, see the SI . The ability of specific pyrone analogues to interfere with biofilm\nformation in B. atropheaus led us to\nexamine the impact on swarming motility. These phenotypes (biofilm\nformation and swarming motility) are linked, with both requiring coordinated\ncommunication between cells and the latter is critical in the initiation\nof the former. 40 Interference with this\nhighly complex behavior would result in a less competitive organism\nat the community level. Pyrones 8 and 9 abolished\nswarming activity in B. atropheaus on\nsemisolid agar, while pyrones 1 and 7 also\nstrongly suppressed swarming motility, although not to the same extent\n( Figure 4 ). Based on\nthe kinetic growth profiles, the absence of swarming motility in the\npresence of 9 could simply be attributed to growth antagonism\nrather than specific interference with the multicellular behavior.\nHowever, the absence of swarming activity in the presence of other\npyrone compounds indicates a more behavioral mechanism.\nThe trend here is similar to that in the antibiofilm test, i.e., pyrones\nwith long alkyl chains at C3 suppress swarming. However, in this case,\nthe naturally occurring PPYA ( 1 ) trended\nwith the analogues. Figure 4 Swarming motility of B. atropheaus with 50 μM compound or carrier control\n(see the SI for details). All data presented\nare the average (±SEM) of at least three independent biological\nreplicates. Statistical analysis was performed by one-way ANOVA with\nBonferroni post hoc corrective testing (* p ≤\n0.05, *** p ≤ 0.001). In terms of biofilm formation, the simpler synthetic pyrones discussed\nso far, possessing a methyl group at C6, showed contrasting effects\non biofilm formation, relative to the naturally occurring PPYA signal (with an i -Bu group at C6). Even compound 5 , which is otherwise identical, gave a dramatically different\nphenotype. Thus, we needed to ascertain the importance of the C6 group.\nFirst, we synthesized the C6- i -Bu compound 24 , without any alkyl group at C3, to examine the properties\nof this analogue ( Scheme 2 ). We then took the best performing C3-alkylated derivatives, 7 , 8 , and 9 , and reacted them with\n2-iodopropane to give compounds 25 (native, PPYC ), 26 (non-native), and 27 (native, PPYE ). Finally, we reversed the positions of the alkyl chains\npresent in PPYA , placing the i -Bu group\nat C3 and the n- hexyl group at C6 ( 28 ). The compounds were then tested for their impact on biofilm formation\nand growth of B. atropheaus as described\nabove. Scheme 2 2-Pyrone Derivatives Synthesized for Further Structure–Activity\nRelationship Studies Method B: (i) TMEDA (1.0 equiv),\nTHF/HMPA (5:1), n -BuLi (2.4 equiv), 0 °C, N 2 and (ii) alkyl iodide (1.8 equiv), 0 °C–rt, 16\nh (see the SI for details). While 1 again led to a notable increase in\nbiofilm\nformation in B. atropheaus , none of\nthe derivative compounds retained this activity ( Figure 5 a). Swarming motility was suppressed\nin the presence of 26 to the same extent as with 1 but was unaffected in the presence of 27 , which\nonly differs by a CH 2 group ( Figure 5 b). This is also consistent with the growth\nkinetics data, and thus, of the pyrone signals and derivative compounds\ntested, 26 was the only one that achieved swarming suppression\nactivity comparable to 1 . However, it should be noted\nthat the growth kinetics of B. atropheaus was affected in the presence of 26 , with the organism\nfailing to reach the growth rate or final biomass achieved in the\npresence of the DMSO control. Compounds 24 , 25 , and 28 led to the abolition of growth on the plate,\nthere was no evidence of colony initiation from the point of inoculation\n( Figure 5 c). Remarkably,\nthe bioactivity of compound 1 toward B.\natropheaus appears to be entirely specific to the\nexact structural arrangement. The activity of the naturally occurring\ncompounds 25 and 27 with respect to cell-clumping\nin Photorhabdus is as yet unknown. 14 The data presented here suggest that they may play a distinct\nsignaling role from the other native pyrones. Figure 5 (a) Biofilm formation\nof B. atropheaus in the presence of derivative\ncompounds 24 – 28 . (b) Swarming motility\nof B. atropheaus in the presence of derivative compounds 24 – 28 . (c) Growth curve analysis of B. atropheaus\nin the presence of derivative compounds 24 – 28 (see the SI for details). All\ndata presented are the average (±SEM) of at least three independent\nbiological replicates. Statistical analysis was performed by one-way\nANOVA with Bonferroni post hoc corrective testing (* p ≤ 0.05, *** p ≤ 0.001)."
} | 4,799 |
23990765 | PMC3749950 | pmc | 6,213 | {
"abstract": "It is generally believed that associative memory in the brain depends on multistable synaptic dynamics, which enable the synapses to maintain their value for extended periods of time. However, multistable dynamics are not restricted to synapses. In particular, the dynamics of some genetic regulatory networks are multistable, raising the possibility that even single cells, in the absence of a nervous system, are capable of learning associations. Here we study a standard genetic regulatory network model with bistable elements and stochastic dynamics. We demonstrate that such a genetic regulatory network model is capable of learning multiple, general, overlapping associations. The capacity of the network, defined as the number of associations that can be simultaneously stored and retrieved, is proportional to the square root of the number of bistable elements in the genetic regulatory network. Moreover, we compute the capacity of a clonal population of cells, such as in a colony of bacteria or a tissue, to store associations. We show that even if the cells do not interact, the capacity of the population to store associations substantially exceeds that of a single cell and is proportional to the number of bistable elements. Thus, we show that even single cells are endowed with the computational power to learn associations, a power that is substantially enhanced when these cells form a population.",
"introduction": "Introduction Associative learning Almost all animals can associate neutral stimuli and stimuli of ecological significance [1] . An extensively studied example is eye-blink conditioning ( Figure 1 ) [2] , [3] . Naïve rabbits respond to an airpuff to the cornea (Unconditioned Stimulus, US) with eyelid closure (Unconditioned Response, UR). By contrast, a weak auditory or visual stimulus (Conditioned Stimulus, CS) does not elicit such an overt response. Repeated pairing of the CS and the US forms a cognitive association between the CS and the US such that the trained animal responds to the CS with eyelid closure, a response known as Conditioned Response (CR). Two important characteristics of associative learning are (1) specificity and (2) generality. The CR does not reflect a general arousal. Rather, the animal learns to respond specifically to the CS. The generality is reflected by the fact that a large family of potential stimuli can serve as a CS if paired with the US. 10.1371/journal.pcbi.1003179.g001 Figure 1 A schematic illustration of eye-blink conditioning. (A) Naïve animal responds to the presentation of an airpuff (the US) by eyelid closure. (B) By contrast, a tone (the CS) does not elicit any overt response. (C) During conditioning the CS and the US are repeatedly paired. (D) After conditioning the animal responds to the CS with eyelid closure (the CR). Neuronal networks are particularly adapted to performing this association and in the last few decades there has been considerable progress in understanding the ways in which experience-based changes in synapses in the nervous system underlie this associative learning process [4] , [5] . Neural network models for associative memory, which explain how both specificity and generality are maintained, are typically based on three elements: (1) Synapses are the physical loci of the memory; (2) synaptic plasticity underlies memory encoding; (3) neural network dynamics, in which the activities of neurons depend on the synaptic efficacies, underlie the retrieval of the learned memories in response to the CS. Genetic regulatory networks Genetic regulatory networks (GRN) describe the interaction of genes in the cell through their RNA and protein products [6] , [7] , [8] . Previous studies have pointed out the similarity between the dynamics of GRNs and the dynamics of neural networks [9] . For example, GRNs, like neural networks, can implement logic-like circuits, where the concentration of a protein (high or low) corresponds to the binary state of the gate [10] , [11] , [12] . These findings prompted us to evaluate the capacity of GRNs to learn associations. Considering associative learning in animals, the US is typically a stimulus of biological significance, such as food or a noxious stimulus that elicits a response (UR) in the naïve animal, either in the form of muscle activation or gland secretion. The GRN correlate of a pain-inducing stimulus is stress. Stressful conditions such as heat, extreme pH, or toxic chemicals often result in a substantial change in the expression level of many different proteins in the cell. For example, Escherichia coli ( E. coli ) bacteria respond to a variety of stress conditions by a general stress response mechanism in which the master regulator controls the expression of many genes [13] . These stressful conditions can be regarded as a US and the resultant change in the expression level of the proteins can be regarded as a UR. By contrast, other stimuli may result in a narrow or absence of a response of the cell and in that sense can be referred to as potential CS. Learning in this framework would correspond to the formation of an association between these potential CS and US such that following the repeated pairing of the CS and US, the presentation of the CS would elicit a UR-like response (CR). The responsiveness of the GRNs to different stimuli has been shown to change over time in response to evolutionary pressure in a manner that resembles associative learning [14] , [15] . These changes take place on time scales that are substantially longer than the lifetime of a single cell and in contrast to associative learning in animals, entail modifications of the genome through mutations. On a shorter timescale, there is some evidence that the single-celled Paramecium can learn to associate a CS with a US within its lifetime [16] . However, these findings have been disputed [17] and the question of whether Paramecia can learn associations and the characteristics of this learning await further experimental validation. The capacity of GRNs to learn associations in shorter, non-evolutionary time-scales has also been studied theoretically using GRN models. Learning in these models is restricted to a small subset of predefined stimuli [18] , [19] , [20] , [21] and thus the computational capabilities of these GRN models are limited compared to neural network models. Here we show that a GRN based on bistable elements and stochastic transitions can learn associations while retaining both specificity and generality. We further compute the capacity of the network and show that the number of different learned associations that the network can simultaneously retain is proportional to the square root of the number of bistable elements. Moreover, this capacity is substantially enhanced when considering a clonal population of GRNs. These results imply that even bacteria are endowed with the capacity to learn multiple associations.",
"discussion": "Discussion In this paper, we explored the ability of a general GRN to encode associations. We showed that a GRN that is endowed with bistable elements and stochastic dynamics is capable of storing and retrieving multiple arbitrary and overlapping associations. The capacity of a single GRN in our model, defined as the number of stored associations, is proportional to the square root of the number of bistable elements . This result is reminiscent of Hopfield-like models with bounded synapses, in which the capacity is proportional to the square root of the number of synapses [47] , [48] , [49] . Remarkably, in a large population of GRNs, as is in a colony of bacteria or in a tissue, this capacity is substantially higher and is proportional to the number of bistable elements. Despite the similarities between the GAM and the Hopfield model, there are two important differences that are noteworthy. First, the capacity of a single GAM may be limited by the presence of readout noise (e.g., in the dynamics of R ). However, this readout noise is not expected to substantially affect the capacity of a population of GAMs because of averaging. Second, the number of neurons available in neuronal networks is much larger than the number of bistable elements in GRNs. Altogether, our model predicts that if the number of bistable elements in the GRN does not exceed several tens, it will be difficult to store more than one or two memories in a single GAM. Therefore, the storage of multiple memories is likely to require a population of GAMs. The key elements in our model are the bistability and the stochasticity of the dynamics of the GRN. Importantly, bistability and stochasticity are not restricted to the transcriptional machinery. Rather, they are found in various cellular processes, including post-transcriptional regulation (e.g., by non-coding RNA [50] , [51] ) or post-translational regulation (e.g. phosphorylation and degradation regulation [36] , [52] , [53] , [54] ). We modeled associative memory that is based on the interaction of proteins through the transcriptional machinery because these dynamics are better characterized and are more accessible experimentally than other cellular alternatives. Moreover, the GAM is not restricted to a particular organism. The parameters used in the simulations presented in this paper are biologically plausible for bacteria. However, because the basic elements of the GAM, namely, bistability and stochasticity, are widespread in GRNs of all cells, the potential for associative learning without a nervous system exists for virtually all cell types, including single-celled eukaryotes and plants. Furthermore, this work suggests that even in animals that possess a nervous system, learning that is independent of this nervous system is also possible. In particular, it could be interesting to consider GAM in the immune system, which has evolved to learn to respond to novel pathogens. Bearing this in mind, we believe that in view of the recent developments of experimental methods that quantitatively measure the expression level of proteins, bacteria, in particular the well characterized E. coli , are the ideal substrate to study the associative learning in GRNs. Each of the components of the GAM module ( Figure 3A ), namely inducible elements, bistable switches and AND gates, have been established in the E. coli transcription network and therefore a synthetic implementation is achievable [55] , [56] . Beyond synthetic implementation, the complexity of the genetic networks suggests that GAM-like modules may exist. A first step in searching for GAMs in known networks should be the identification of plausible candidates for the US, UR and CS. In animals, the US is a stimulus that causes an overt response prior to learning, the UR. Typically the US is a stimulus of biological significance, such as food or a noxious stimulus and the UR is an ecologically-relevant overt response, often in the form of muscle activation. For example, in the eye-blink conditioning experiment ( Figure 1 ) the US is an air puff and the UR is an eye blink that protects the eye from the puff. An important point to consider when searching for associative learning in bacteria is ecological significance. Our model for associative learning, similar to models of associative learning in neuronal networks, does not incorporate any ecological information about the stimuli. However in animals, it is known that the ability to form an association depends on the ecological relevance of the CS to the US. For example, the association of the taste of a certain food (CS) with the symptoms caused by a toxic or spoiled food (US), known as taste aversion, is easily-formed after a small number of repetitions. By contrast, it is substantially more difficult to form an association of a tone with the same US [57] . It is generally believed that this difference results from the fact that typically, taste is more informative about the chemical composition of substances than auditory signals. Therefore, taste-aversion but not tone-aversion has evolved as a specific learning mechanism aimed at preventing the consumption of poisonous substances. Drawing an analogy to associative learning in bacteria, we propose to utilize ecologically-relevant CS rather than arbitrary CS when searching for associative learning in bacteria. In our model, the strength of association increases with the number of repetitions due to the stochasticity in the encoding process. Such dependence of the strength of association on the number of repetitions is also observed in classical conditioning experiments in animals [58] . Therefore, experiments involving a large number of co-occurrences of the CS and US are more likely to reveal associative learning in GRNs or populations of GRNs. Note that standard experiments studying responses of bacteria are typically short and do not involve repetitions in the presentation of stimuli to the same population of bacteria. Therefore, associative learning in such experiments may have been overlooked. Moreover, we have shown that the learning capacity of the population of bacteria is higher than that of a single GRN. Therefore, the experimental search for associative learning in bacteria should be done at the population level. More specifically in bacteria, the presence of foreign bacteria is a signal of potential stress. For example, many bacteria produce antibiotics that are harmful to other strains [59] . Other bacteria are sensitive to these damaging antibiotics and respond to their presence by activating a pre-wired stress response, such as the multiple antibiotics response (MAR) [60] . We thus suggest that the R gene in our scheme corresponds to one of the outputs of MAR response, e.g. the micF gene [61] . Note that similar to the blink in the classic eye-blink conditioning that protects the eye from the air puff ( Figure 1 ), the activation of micF prevents the entry of the antibiotics into the cell. Thus, the antibiotics can be considered as a US whereas the stress response can be considered as a UR. However, the production of harmful antibiotics is not present in all bacteria species. Therefore, learning to distinguish between harmful and benign strains of bacteria is of potential great ecological significance because it may allow the bacteria to respond faster. Thus, the presence of foreign bacteria could correspond to the CS in our framework. Indeed, bacteria are able to detect secondary metabolites that are produced by other strains [62] . In that line, we suggest as a candidate for the M protein in the model the MarA gene. MarA is known to positively autoregulate itself, and thus has the potential to be bistable. In addition, the promoter of that gene contains multiple binding sites for transcription factors, allowing for complex regulation of the gene expression including the realization of AND gates. Experimentally, the UR can be measured using a fluorescent-based reporter that is regulated by a promoter of a stress response gene. The CS in this framework should be stimuli that can be sensed by the bacteria but do not elicit the stress response. These include a change in the concentration of different molecules that does not activate the stress response. Repeated exposure to such conditions can be controlled using a chemostat [63] , which can maintain selected growth conditions at a constant level while changing others. Finally, the benefit of the stress response at the population level can also be found in the induction of the MAR response, as it triggers the activation of genes that inactivate toxic compounds. The benefit of this “pooled response” for the population comes from the decrease in the concentration of the toxic compound [64] . Whether or not associative learning exists in GRNs on a time-scale much shorter than required for evolution is an open question. However, whether considering bacteria that can predict a stress condition or human digestive cells that can predict food intake, associative learning in single and populations of cells seems to have an evolutionary advantage. In view of the computational capabilities of GRNs demonstrated in this paper, we believe that future careful investigations will reveal the existence of associative learning in single and populations of cells."
} | 4,079 |
35548749 | PMC9085637 | pmc | 6,215 | {
"abstract": "Plant growth-promoting rhizobacteria (PGPR) not only promote growth and heavy metal uptake by plants but are promising biosorbents for heavy metals remediation. However, there exist arguments over whether extracellular adsorption (biosorption) or intracellular accumulation (bioaccumulation) play dominant roles in Cd( ii ) adsorption. Therefore, three cadmium-resistant PGPR, Cupriavidus necator GX_5, Sphingomonas sp. GX_15, and Curtobacterium sp. GX_31 were used to study bioaccumulation and biosorption mechanisms under different initial Cd( ii ) concentrations, using batch adsorption experiments, desorption experiments, scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX) spectroscopy, transmission electron microscopy (TEM), and Fourier-transform infrared (FTIR) spectroscopy. In this study, with the increase of the initial Cd( ii ) concentrations, the removal efficiency of strains decreased and the adsorption capacity improved. The highest Cd( ii ) removal efficiency values were 25.05%, 53.88%, and 86.06% for GX_5, GX_15, and GX_31 with 20 mg l −1 of Cd( ii ), while the maximum adsorption capacity values were 7.97, 17.13, and 26.43 mg g −1 of GX_5, GX_15, and GX_31 with 100 mg l −1 of Cd( ii ). Meanwhile, the removal efficiency and adsorption capacity could be ordered as GX_31 > GX_15 > GX_5. The dominant adsorption mechanism for GX_5 was bioaccumulation (50.66–60.38%), while the dominant mechanisms for GX_15 and GX_31 were biosorptions (60.29–64.89% and 75.93–79.45%, respectively). The bioaccumulation and biosorption mechanisms were verified by SEM-EDX, TEM and FTIR spectroscopy. These investigations could provide a more comprehensive understanding of metal-bacteria sorption reactions as well as practical application in remediation of heavy metals.",
"conclusion": "4. Conclusions Three cadmium-resistant PGPR, Cupriavidus necator GX_5, Sphingomonas sp. GX_15, and Curtobacterium sp. GX_31, were used to study bioaccumulation and biosorption mechanisms under different initial Cd( ii ) concentrations. Removal efficiency and adsorption capacity of the assessed PGPR can be ordered as GX_31 > GX_15 > GX_5. Strain GX_15 showed high potential (86.06%) for Cd( ii ) remediation. Physical entrapment, ion exchange, and complexation were involved in biosorption processes. The dominant adsorption mechanism for GX_5 was bioaccumulation, while the dominant mechanisms for GX_15 and GX_31 were both biosorptions. The elucidation of the binding mechanisms could provide new perspectives of strains in practical bioremediation applications for heavy metals. However, more strains from different genera or even phyla are needed to be assessed for biosorption and bioaccumulation mechanisms under different metal concentrations and using various analysis methods.",
"introduction": "1. Introduction Remediation of heavy metal-contaminated soil has received much attention due to heavy metals' adverse effects on plants, animals, microorganisms, and humans. Among hazardous metals, cadmium (Cd) is of particular concern because it is difficult to degrade, accumulates easily, and is highly toxic. 1 It has been demonstrated that a small amount of Cd( ii ) in the food chain can cause health risks in humans. 2 Physicochemical approaches such as filtration, ion exchange, chemical precipitation, and solvent extraction are widely used to remove heavy metals from the environment. 3 However, these applications are mostly ineffective, expensive, and nonspecific, especially when concentrations of heavy metals are low. 4 Therefore, it is imperative to find an efficient, cost-effective alternative. The use of microbiological biomass, including bacteria, 5 fungi, 6,7 and yeast, 8 is increasingly accepted in metals removal due to the large and well defined surface area of biomass, its high binding affinity, its environmental friendliness, and its low cost. 9 Microbial remediation takes place mainly through biosorption or bioaccumulation mechanisms. 10 Biosorption is a passive-process, metabolism-independent extracellular adsorption, where heavy metal ions are passively adsorbed onto components of the cell surface. 11 Generally, biosorption contains the following mechanisms: physical entrapment (physical adsorption), ion exchange, and complexation in functional groups, 12,13 which may be independently or synergistically involved. 14 Bioaccumulation, on the other hand, is an active-process, metabolism-dependent intracellular accumulation. 15 It is a more complex process entailing many occurrences, including localization of the metal within specific organelles, metallothionein binding, and efflux pumping. 14,16 Microorganisms show promise for the removal of heavy metals from polluted environments through both bioaccumulation and biosorption processes. Bioaccumulation and biosorption have been extensively studied by some researchers. 17–19 However, there have been arguments on whether bioaccumulation or biosorption plays a dominant role in Cd( ii ) adsorption. 20–22 Little work has been done to investigate the predominant mechanisms (bioaccumulation and biosorption) involved in the reduction of Cd( ii ) toxicity of Gram-positive and Gram-negative bacteria at a time. Moreover, many researchers did not take metal concentrations into consideration during the adsorption process. 23,24 The objectives of the present work were: (1) to investigate the capacities of Cd( ii )-resistant PGPR, i.e. , Cupriavidus necator GX_5 (CP002878), Sphingomonas sp. GX_15 (MF959440), and Curtobacterium sp. GX_31 (MF959445), for Cd( ii ) adsorption under the same experimental conditions; (2) to analyze surface interaction between static biomass and Cd( ii ) by means of SEM-EDX, TEM, and FTIR analysis; (3) to elucidate the main adsorption mechanism (bioaccumulation or biosorption) of bacteria for Cd( ii ) under different initial Cd( ii ) concentrations using batch adsorption experiments; and (4) to provide new insight into Cd-resistant PGPR's potential use for bioremediation of contaminated environments.",
"discussion": "3. Results and discussion 3.1. Cd( ii ) adsorption by biosorbent Under optimal conditions (pH: 6.0; reaction time: 6 h; biomass dosage: 1.0 g l −1 ) based on the preliminary data, the batch adsorption experiments were conducted with initial Cd( ii ) concentrations: 20, 50, and 100 mg l −1 . The largest Cd( ii ) removal efficiency was 86.06%, for Curtobacterium sp. GX_31 under the initial concentration of 20 mg l −1 ; while the minimal removal efficiency was 7.98%, for Cupriavidus necator GX_5 with 100 mg l −1 Cd( ii ) treatment. It was obviously observed that a significant difference in Cd( ii ) adsorption existed among the three strains—more specifically, GX_31 > GX_15 > GX_5 ( p < 0.05)—given the same amount of Cd( ii ) ( Fig. 1A ). In the study, biomass dosage (1.0 g l −1 ) was measured by weighing the freeze-dried fine powder of biosorbent, which was time-consuming but more accurate than methods in some previous studies. 28–30 Meanwhile, the batch adsorption experiments were conducted using resting biomass rather than growing cells in medium. Considering the different growth rates of strains in medium, it is inappropriate to compare adsorption efficiency and capacity between strains. However, it makes sense that removal efficiency varied among strains based on their own adsorption or defence mechanisms. 10 Fig. 1 The removal efficiency of Cd( ii ) (A) and adsorption capacity of Cd( ii ) (B) by Cupriavidus necator GX_5, Sphingomonas sp. GX_15, and Curtobacterium sp. GX_31 under 20, 50, and 100 mg l −1 of initial Cd( ii ) concentrations. In Fig. 1A , we also see that removal efficiency was significantly higher at lower concentrations than at higher concentrations for the same strains, which is in accordance with other studies. 15,29,31 At low concentrations, the ratio of the moles of Cd( ii ) to the available surface area was low, leaving a large number of binding sites free for Cd( ii ) interactions and resulting in high adsorption efficiency. 32 On the contrary, at high concentrations of Cd( ii ), a lack of sufficient free binding sites resulted in low removal efficiency. 33 Similarly, the three strains showed a range of adsorption capacities when treated under the same Cd( ii ) concentrations: GX_31 > GX_15 > GX_5 ( p < 0.05) ( Fig. 1B ). In considering identical strains, adsorption capacity was stronger at higher concentrations than that at lower concentrations ( Fig. 1B ). The strongest and weakest adsorption capacities were 26.43 mg g −1 for GX_31 at 100 mg l −1 of Cd( ii ) and 5.01 mg g −1 for GX_5 at 20 mg l −1 , respectively. High initial concentrations could provide an effective force for driving metal ions to interact with finite metal binding sites, prompting adsorption by biomass strains. 34 However, the adsorption capacity of the biosorbent would reach a saturation value with the increase of initial metal concentrations due to limited binding sites. 26 3.2. Desorption of Cd( ii ) from loaded cell biosorbent The amounts of Cd( ii ) desorbed from GX_5, GX_15, and GX_31 by water, NH 4 NO 3 , and EDTA-Na 2 were investigated and displayed in Fig. 2 , which shows that 5.67–8.95% and 0.97–3.93% of Cd( ii ) adsorbed by GX_15 and GX_31, respectively, was desorbed by water; 56.09–59.66% and 71.20–75.70%, respectively, was desorbed by NH 4 NO 3 ; and 60.29–64.89% and 75.93–79.45% by EDTA-Na 2 ( Fig. 2B and C ). In comparison, 21.21–30.34% of Cd( ii ) adsorbed by GX_5 was desorbed by water, while 37.48–43.58% and 39.62–49.34% was desorbed by NH 4 NO 3 and EDTA-Na 2 , respectively ( Fig. 2A ). Fig. 2 The percentage of Cd( ii ) desorbed from Cd( ii )-loaded biomass of Cupriavidus necator GX_5 (A), Sphingomonas sp. GX_15 (B), and Curtobacterium sp. GX_31 (C), after treatment with ddH 2 O, 1.0 mol l −1 of NH 4 NO 3 , and 0.1 mol l −1 of EDTA-Na 2 , under 20, 50, and 100 mg l −1 initial Cd( ii ) concentrations. These results indicated that of the Cd( ii ) adsorbed by GX_5, 21.21–30.34% was physically entrapped, 7.82–16.27% was held by ion exchange, 2.14–5.76% was complexed in functional groups ( Fig. 2A ), and 50.66–60.38% was accumulated inside the cells ( Fig. 3 ). On the contrary, for GX_15 and GX_31, 5.67–8.95% and 0.97–3.93% was physically entrapped. This suggested that the bound Cd( ii ) was not easily released and the contribution of physical adsorption was minor. According to Fang et al. , only 3.6% of Cd( ii ) was adsorbed physically by Spirulina sp. 23 A similar study was conducted by Chojnacka et al. , who pointed out that the maximum contribution of physical adsorption by the blue-green algae Spirulina sp. was 3.7%. 35 Desorption rates for Cd( ii ) held by ion exchange for strains GX_15 and GX_31 were 47.14–53.81% and 67.28–74.73%, respectively; rates for Cd( ii ) complexed in functional groups were 4.20–5.41% and 3.75–4.73%, respectively ( Fig. 2B and C ); 35.11–39.71% of Cd( ii ) was bioaccumulated in GX_5 and 20.55–24.07% in GX_15 ( Fig. 3 ). Fig. 3 Extracellular adsorption (biosorption) and intracellular accumulation (bioaccumulation) by Cupriavidus necator GX_5, Sphingomonas sp. GX_15, and Curtobacterium sp. GX_31 under 20, 50, and 100 mg l −1 initial Cd( ii ) concentrations. As these figures show, regardless of initial Cd( ii ) concentrations, the dominant mechanism for Cd( ii ) adsorption was bioaccumulation (intercellular accumulation) (50.66–60.38%) for GX_5, while the dominant adsorption mechanisms were both biosorptions (extracellular adsorptions) (60.29–64.89% for GX_15 and 75.93–79.45% for GX_31) ( Fig. 3 ). It is obvious that the biosorption mechanism of GX_5 is more prone to physical entrapment (21.20–30.33%), while those of GX_15 and GX_31 tend toward ion exchange (47.14–53.81% and 67.28–74.73%, respectively). Adsorption mechanisms differed due to varying compositions and structures in bacterial cell walls. 25 Another reason for adsorption mechanisms differing might be that exclusion mechanisms lead to Cd( ii ) being excreted from inside the cell and improving surface binding via metal-exporting proteins. 36 Some researchers have concluded that the increase in surface adsorption might be a result of extracellular polymeric substances protecting cells from Cd( ii ) toxicity. 21,37 The result was different from that obtained by Huang et al. , who illustrated that intracellular accumulation is the main adsorption mechanism given lower metal concentrations and extracellular adsorption is the main adsorption mechanism at higher concentrations. 11 Due to variance in experimental conditions and analytical methods, it is inappropriate to compare adsorption mechanisms between researchers. 38–41 However, although study results may not be directly comparable, we can be sure that strains GX_5, GX_15, and GX_31 show different Cd( ii ) adsorption mechanisms and capacities. Meanwhile, bioaccumulation and biosorption were verified by SEM-EDX, TEM, and FTIR spectroscopy, which will be discussed in the following sections. 3.3. SEM-EDX and TEM analysis To improve understanding of the mechanisms of Cd( ii ) interactions with microbes, SEM-EDX and TEM were performed. Cell surfaces of GX_5, GX_15, and GX_31 were all observed to be rod shapes with clear boundary before adsorption ( Fig. 4A-a, B-a and C-a ). There were no obvious changes in morphology of these strains after interaction with Cd( ii ) at a concentration of 20 mg l −1 ( Fig. 4A-b, B-b and C-b ). However, their surfaces became rough and were covered by sediments after the reaction; this effect was yet more evident when bacterial cells were exposed to 100 mg l −1 Cd( ii ) ( Fig. 4A-d, B-d and C-d ). Changes in cell morphology could be explained as a protective mechanism responding to a stressful environment, which has previously been reported. 42,43 Moreover, for strain GX_15, cells appeared to aggregate after reaction with Cd( ii ), an effect which Fig. 4B-c and B-d show to be especially pronounced. Some floccus precipitation was found on the surface of GX_15 ( Fig. 4B-b, B-c and B-d ). Aggregation and precipitation might be caused by extracellular polymeric substances, which had an important role in binding heavy metals. 30 EDX is a useful tool for chemical and elemental analysis of biosorbents and has been extensively applied. 44 EDX spectra recorded the signals of carbon, nitrogen, and oxygen, which were likely in polysaccharides and proteins of the biosorbents (Fig. S1 † ). Unloaded biomass showed no Cd( ii ) signals in the EDX spectra, but signals could be observed after Cd( ii ) exposure, revealing the presence of Cd( ii ) in the cell after adsorption (Fig. S1 † ). However, EDX spectra could only determine the presence or absence of Cd( ii ) on the biomass qualitatively, not quantitatively. Fig. 4 SEM images of Cupriavidus necator GX_5 (A), Sphingomonas sp. GX_15 (B), and Curtobacterium sp. GX_31 (C) under different initial Cd( ii ) concentrations ((a) 0 mg l −1 of Cd( ii ); (b) 20 mg l −1 of Cd( ii ); (c) 50 mg l −1 of Cd( ii ); and (d) 100 mg l −1 of Cd( ii )). Although most heavy metals are not essential to bacteria, some of them can cross the cell membrane and enter the cells via a range of processes. 45 Therefore, TEM analysis of strains was conducted to intuitively show the effects of metal concentrations on cells. As shown in Fig. 5A-a, B-a and C-a , the cells were intact, the contents were identically dispersed in the cells, and the cell wall could be clearly distinguished from cytoplasm. With increased initial Cd( ii ) concentrations, the cell walls became unclear and vague and it was hard to tell the cell wall from cytoplasm ( Fig. 5A-b, A-c, B-b, B-c, C-b and C-c ). This phenomenon was especially evident given a Cd( ii ) concentration of 100 mg l −1 ( Fig. 5A-d, B-d and C-d ). Fig. 5B-c and B-d show that, under concentrations of 50 and 100 mg l −1 of Cd( ii ), some contents flowed out of the cells. This indicates that the cell walls of GX_15 were destroyed by high Cd( ii ) concentrations, and thus that GX_15 is more sensitive to Cd( ii ) than GX_5 and GX_31. Fig. 5 TEM images of Cupriavidus necator GX_5 (A), Sphingomonas sp. GX_15 (B), and Curtobacterium sp. GX_31 (C) under different initial Cd( ii ) concentrations ((a) 0 mg l −1 of Cd( ii ); (b) 20 mg l −1 of Cd( ii ); (c) 50 mg l −1 of Cd( ii ); and (d) 100 mg l −1 of Cd( ii )). 3.4. FTIR spectra study To investigate possible interactions between Cd( ii ) and functional groups on the cell walls, the FTIR spectra of GX_5, GX_15, and GX_31 were recorded before and after Cd( ii ) adsorption. The pre-adsorption FTIR spectra revealed the presence of many functional groups on the cell surface, indicating the complex nature of the strains ( Fig. 6A-a, B-a and C-a ). Meanwhile, the IR spectra of GX_15 and GX_31 were similar to each other, but different from that of GX_5 ( Fig. 6A–C ). Fig. 6 FTIR images of Cupriavidus necator GX_5 (A), Sphingomonas sp. GX_15 (B), and Curtobacterium sp. GX_31 (C) under different initial Cd( ii ) concentrations ((a) 0 mg l −1 of Cd( ii ); (b) 20 mg l −1 of Cd( ii ); (c) 50 mg l −1 of Cd( ii ); and (d) 100 mg l −1 of Cd( ii )). Broad spectra bands were observed in the range of 3300–3500 cm −1 , representing the stretching bond of the –NH from an amino group and a bonded hydroxyl group. 46 After contact with Cd( ii ), the spectra had the tendency to shift to lower frequencies ( Fig. 6A–C ), an effect that was more evident for GX_31 ( Fig. 6C ). The band around 2930 cm −1 corresponded to symmetrical –CH– vibration of –CH 2 and –CH 3 in lipids, 47 which showed subtle changes after adsorption ( Fig. 6 ). For strains GX_5 and GX_15, there were no changes at the band of 2850 cm −1 , which corresponded to asymmetrical –CH– vibration in lipids. 34 However, the adsorption peaks at 2850 cm −1 for GX_31 shifted from 2847.50 cm −1 to 2850.38 cm −1 (20 mg l −1 Cd( ii )-loaded), 2851.41 cm −1 (50 mg l −1 Cd( ii )-loaded), and 2851.43 cm −1 (100 mg l −1 Cd( ii )-loaded) ( Fig. 6C ). Carbonyl groups stretching vibration was prominent at 1741.17 cm −1 for strain GX_5 ( Fig. 6A ). 48 After interaction with 20, 50, and 100 mg l −1 concentrations of Cd( ii ), the spectra of Cd( ii )-loaded biomass demonstrated a clear shift of this peak to 1726.90 cm −1 , 1727.02 cm −1 , and 1728.50 cm −1 , respectively. Strains GX_15 and GX_31 did not show any peak at this band ( Fig. 6B and C ). The peaks between 1650 cm −1 and 1540 cm −1 could be assigned to amide groups in proteins. 49 The typical amide I (–CO–) appeared at 1655.52 cm −1 , 1653.55 cm −1 , and 1655.88 cm −1 , respectively, for GX_5, GX_15 and GX_31; while the peaks at 1543.01 cm −1 , 1543.59 cm −1 , and 1545.35 cm −1 were considered to be amide II (–NH–). The spectra showed a minor shift of these two bands to 1654.58 cm −1 and 1655.47 cm −1 for GX_5 and 1542.15 cm −1 and 1544.79 cm −1 for GX_31 ( Fig. 6A and C ); for GX_15, the two bands shifted to 1656.25 cm −1 and 1537.34 cm −1 ( Fig. 6B ). A minor peak shift at 1397.42 cm −1 to 1398.12 cm −1 for GX_15 ( Fig. 6B ), 1399.15 cm −1 to 1402.46 cm −1 for GX_31 ( Fig. 6C ), and 1382.24 cm −1 to 1381.33 cm −1 for GX_5 ( Fig. 6A ) indicated the role of carboxyl groups in Cd( ii ) binding. 31,50 For GX_15, there existed a significant shift from 1082.43 cm −1 to 1065.38 cm −1 , corresponding to the –CO– group vibration in the cyclic structure of carbohydrates. 26 Meanwhile, a band at 1070.29 cm −1 shifted to 1067.82 cm −1 for GX_31, representing the –CO– groups as well. 20 In the control spectra, the adsorption peak at 1056.64 cm −1 due to the phosphate groups was observed, 38 and a shift of this peak to 1053.76 cm −1 (50 and 100 mg l −1 Cd( ii )-loaded) ( Fig. 6A ) suggested the interaction of bound metals with phosphates. After Cd( ii ) adsorption occurred, the overall IR spectra analysis indicated the involvement of functional groups such as hydroxyl, carbonyl, and carboxyl groups of saccharides; amino and amide groups of proteins; phosphate groups; and –COC– groups of carbohydrates in the interaction of Cd( ii ) with bacteria. Moreover, with increased initial metal concentrations, the differences between IR spectra for Cd( ii )-free and for Cd( ii )-loaded cells was more distinct."
} | 5,096 |
34026006 | PMC8131813 | pmc | 6,216 | {
"abstract": "Abstract \n Ectomycorrhizal (ECM) symbiosis is an evolutionary biological trait of higher plants for effective nutrient uptakes. However, little is known that how the formation and morphological differentiations of ECM roots mediate the nutrients of below‐ and aboveground plant tissues and the balance among nutrient elements across environmental gradients. Here, we investigated the effects of ECM foraging strategies on root and foliar N and P concentrations and N:P ratio Abies faxoniana under variations of climate and soil conditions. The ECM symbionts preferentially mediated P uptake under both N and P limitations. The uptake efficiency of N and P was primarily associated with the ECM root traits, for example, ECM root tip density, superficial area of ECM root tips, and the ratio of living to dead root tips, and was affected by the ECM proliferations and morphological differentiations. The tissue N and P concentrations were positively associated with the abundance of the contact exploration type and negatively with that of the short‐distance exploration type. Our findings indicate that the nutritional status of both below‐ and aboveground plant tissues can be strongly affected by ECM symbiosis in natural environments. Variations in the ECM strategies in response to varying environmental conditions significantly influence plant nutrient uptakes and trade‐offs.",
"introduction": "1 INTRODUCTION Over 80% of tree species form ectomycorrhizal (ECM) symbionts, which are essential for maintenance of forest ecosystem health and effective soil nutrient uptakes by host trees (Barrett et al., 2011 ; Smith & Read, 2008 ). The ECM roots facilitate soil nutrient uptake through branching root tips and emanating hyphae. The ECM‐infested roots vary greatly in shape and structural configurations (Agerer, 1987–2006 ; Agerer, 1991 ) and are functionally differentiated in the capacity of soil exploration range. Agerer ( 2001 ) classified the ECM roots into five types based on the extent of soil exploration: the contact exploration (smooth mantle and only a few emanating hyphae), the short‐distance exploration (ECM root with a voluminous envelope of emanating hyphae but no rhizomorphs), the medium‐distance exploration (ECM root with rhizomorphs), the long‐distance exploration (ECM root with long rhizomorphs), and the pick‐a‐back exploration (ECM formed by members of the Gomphidiaceae ). The five ECM exploration types differ in their capability of reaching out for soil resources at distances from the root tips through variations in the length of emanates (Pritsch & Garbaye, 2011 ; Tedersoo et al., 2012 ). The types and morphologies of ECM are found to respond to variations in soil and climatic conditions (Graefe et al., 2010 ; Ostonen et al., 2009 ; Rosinger et al., 2018 ; Toljander et al., 2006 ), and greatly affect the nutrient uptake capacity and efficiency of the host trees (Chen et al., 2016 , 2018 ). Both N and P are essential nutrient elements for plants and other organisms, but their limitations are common in terrestrial ecosystems. The uptake efficiency of N and P is dependent on the root systems with strategies adapting to environmental conditions (Chien et al., 2011 ; Hodge, 2004 ; Jackson & Caldwell, 1996 ). Of which, the ECM symbionts play an important role when trees undergo environmental stresses (Ahonen‐Jonnarth et al., 2000 ; Alonso et al., 2003 ) or nutrient deficiency (Almeida et al., 2019 ; Hajong et al., 2013 ). The symbionts help improve soil nutrient absorption by altering hyphae length, modifying the morphologies of root tips, or affecting microbial communities, when trees are under stresses (Boomsma & Vyn, 2008 ; Lõhmus et al., 2006 ; Ostonen et al., 2009 ). Under natural environmental conditions, uptakes of N and P by roots are enhanced by ECM foraging strategies in favor of extended exploration of soil resources (Ostonen et al., 2007 , 2011 ). The important foraging strategies discovered so far include the secretion of enzymes decomposing N or P complex by ECM root tips, and facilitation of nutrient acquisition far from the root distal by extending hyphae or rhizomorphs (Courty et al., 2010 ; Nehls & Plassard, 2018 ; Pritsch & Garbaye, 2011 ). ECM plants are characteristically of low foliar nutrients and high leaf mass per unit area, especially the tree species in Pinaceae and Fagaceae families (Cornelissen et al., 2001 ; Koele et al., 2012 ; Read, 1991 ). The intimate connections of foliar N nutrition and ECM symbiosis are widely reported (Hobbie et al., 2005 ; Hobbie & Hobbie, 2006 ; Koele et al., 2012 ). For instance, isotope tracing experiments provided direct evidence of the N transfers among plant tissues and mycorrhizal fungi (Hobbie & Högberg, 2012 ; Steven et al., 2004 ). Still, few studies have reported the associations between ECM traits and foliar nutrients. There are observations of the associations of root and leaf nutrient traits (Craine & Lee, 2003 ; Tjoelker et al., 2005 ) and reports of the positive correlations of N or P between roots and leaves (Güsewell, 2004 ; Liu et al., 2010 ). The mycorrhizal root systems are known to have the capability of assimilating N and P and then transferring them to shoots (Michelsen et al., 1996 ; Plassard & Dell, 2010 ; Smith & Read, 2008 ). Previous research has revealed the relationship of foliar N with mycorrhizal fungi, asserting that mycorrhizal associations influence the foliar N transfer (Craine et al., 2009 ; Hobbie & Hobbie, 2006 ). Controlled experiments demonstrated that the mycorrhizal symbionts affected the allocation of N and P nutrients among roots, stems, and leaves (Brandes et al., 1998 ; Chen et al., 2010 ; Johnson, 2010 ; Landis & Fraser, 2008 ; Wang et al., 2006 ). However, how ECM strategies mediate the below‐ and aboveground nutrients balance in plants in response to environmental changes yet remains unelucidated. \n Abies faxoniana is an ancient species in the genus Abies that experienced the glacial and interglacial periods (Florin, 1963 ). It is a typical ECM tree species and naturally distributed from 2,700 to 3,900 m asl . in subalpine area of Sichuan Province, Southwest China. A. faxoniana forest is the primary vegetation type in that subalpine ecosystem. In this study, we investigated the effects of ECM strategies on the N and P nutrient uptake between below‐ and aboveground tissues in plants under different environmental gradients, that is, varying mean annual temperatures, mean annual rainfall, elevations, and soil types. The root and foliar N and P contents, ECM traits representing nutrient uptake pathway, and efficiency were measured. Our objective was to determine how the ECM strategies in A. faxoniana regulated the nutrient preference of N and P nutrition in below‐ and aboveground tissues. We hypothesized that (a) ECM strategies mediate the partiality of N and P nutrition in below‐ and aboveground tissues in A. faxoniana in response to environmental variations, and (b) ECM soil exploration types differentially regulate the nutrient uptakes in host trees.",
"discussion": "4 DISCUSSION It is widely recognized that ECM fungi promote the uptake of N and P in plants. While the root N and P nutrition of ECM plants has been widely studied (Almeida et al., 2019 ; Franklin et al., 2014 ; Zhang et al., 2019 ), relatively few studies have attempted to determine the attributions of ECM symbionts to aboveground nutrition in tree species (Koele et al., 2012 ; Michelsen et al., 1996 ). In this study, we examined the effects of the variations in ECM symbiosis on root and foliar N and P nutrition in A. faxoniana under varying soil types and climate factors. Generally, the ECM in A. faxoniana appeared to be more important in P uptake than N uptake under both N and P limitations (Figures 1 , 2 , and 5 ). The ECM traits in A. faxoniana were better correlated with root N and P concentrations than with the foliar N and P concentrations (Figure 2 ). The ECM soil exploration types exerted differential impacts on root and foliar N concentrations and N:P ratio (Figure 4 ). FIGURE 5 A conceptual model of the intervention of ECM symbiosis on root and foliar N and P in Abies faxoniana . I: The primary effects of ECM symbiosis on root nutrients. Root N and P nutrients are both strongly affected by ECM symbiosis, but the effects are stronger on root P than root N; II: indirect mediation of ECM symbiosis on foliar N and P driven by the nutrient limitation signals from leaves to roots; III: changes in foliar N and P caused by variations in ECM strategies. Changes in ECM foraging strategies impose greater influences on foliar P than on foliar N 4.1 Differential effects of ectomycorrhizal strategies on the below‐ and aboveground plant N and P nutrients Concerning our first hypothesis that ECM strategies mediate the partiality of N and P nutrition in below‐ and aboveground tissues in A. faxoniana in response to environmental variations, we found distinct effects of ECM strategies on plant N and P elemental stoichiometry in roots and leaves under varying soil and climate conditions. In this study, the mature A. faxoniana trees were deficient in root P (Table 1 , P concentration: 0.72 ± 0.13 mg/g) as well as in root and foliar N (values of N concentration < 10 mg/g and N:P ratio < 14). It is suggested that the values of N:P ratio < 14 or >16, respectively, indicate N limitation or P limitation in plants and that tissue P concentration < 1 mg/g and tissue N concentration < 10 mg/g are considered as deficient of the nutrient (Güsewell, 2004 ; Güsewell & Koerselman, 2002 ; Tessier & Raynal, 2003 ). P is generally more limiting than N in terrestrial ecosystems as it is derived primarily from rock weathering and uniquely depended on root systems (Vitousek et al., 2010 ; Walker & Syers, 1976 ). According to the results in this study, the variations of ECM traits in A. faxoniana affected more on root and foliar P concentrations than on N concentrations (Figures 1 and 2 ), suggesting that ECM strategies are more functional on P uptakes than on N uptakes under both N and P limitations. Basically, the resource allocation in belowground by the mycorrhizal symbiosis is expected to abide by the nutrient requirements of plants (Merrild et al., 2013 ). However, the priority in nutrient acquisition is frequently determined by the strategic choices of plant species under multiple element limitations. It has been demonstrated that the ECM symbiosis give priority to the uptake of P but not N when in deficient supplies under different experimental conditions (Almeida et al., 2019 ; Smith et al., 2011 ; Zavišić et al., 2016 ). Moreover, it has been reported that the ECM symbioses sometimes do not largely alleviate N limitation (Franklin et al., 2014 ; Näsholm et al., 2013 ) and that plants could obtain N by the root pathway rather than the mycorrhizal symbioses which would require extra C investment under N shortage (Jiang et al., 2017 ; Zhang et al., 2019 ). The differential mechanisms of nutrient acquisition might change the nutrition preferences in plant species (Houlton et al., 2007 ; Zhang et al., 2018 ). Apart from soil resources and climate factors, our study shows that the varied ECM traits greatly influenced N and P nutrients in A. faxoniana (Figures 1 and 2 ). Overall, the ECM traits associated with the uptake efficiency, such as the colonization ratio of ECM root tips, the ratio of the living to dead root tips, the colonization ratio of the contact exploration type, and the superficial area of ECM root tips, were all positively correlated to the below‐ and aboveground N and P concentrations in A. faxoniana (Figure 3 ). However, the fine root biomass and morphological diversity of ECM roots impacted negatively on the tissue N and P concentrations but positively on the N:P ratio, suggesting the trade‐offs between the C investment for ECM root proliferation and morphology differentiation and the nutrients uptake. Accordingly, we draw the conclusion that both N and P nutrients of roots and leaves in A. faxoniana are primarily mediated by the nutrient uptake efficiency of ECM roots, while the N and P stoichiometry is strongly related to the alteration of uptake or transportation pathway of ECM roots. Research shows that the nutrient uptake efficiency of the symbiotic fungi in plants might mediate the concentration of the nutrients in roots and leaves, for example, the ECM colonization ratio, ECM absorption root vigor (Beltrano et al., 2013 ; Li et al., 2015 ; Vandenkoornhuyse et al., 2003 ), ECM root tip density, and absorptive capacity of ECM emanates (Ostonen et al., 2011 ), while the N and P stoichiometry in plant species could be affected by the function of mycorrhizal symbionts, for example, hyphae exploration ability and/or extracellular enzyme secretion (Chen et al., 2010 ). Plant nutrient uptake and balance exceedingly depend on the alternative foraging strategies of the ECM root systems (e.g., foraging precision of hyphae, morphology plasticity, and foraging range) under different environmental conditions (Chen et al., 2018 ; Einsmann et al., 1999 ; Köhle et al., 2018 ; Wang et al., 2006 ). While controlled experiments and isotope tracing studies have demonstrated that ECM symbionts contribute to the improvements of plant biomass, foliar N and P acquisition (Brandes et al., 1998 ; Craine et al., 2009 ; Hobbie & Hobbie, 2006 ), such functional roles are not readily observable in natural ecosystems due to the confounding effects of biotic and biotic environments. In this study, under the varying soil and climate conditions, we were able to reveal the differential roles of ECM strategies in N and P uptake in A. faxoniana . 4.2 Trade‐offs of nutrient uptake and soil exploration types in Abies faxoniana \n In confirmation to our second hypothesis that ECM soil exploration types differentially regulate the nutrient uptakes in host trees, we found that the concentrations of root and foliar N and P in A. faxoniana were positively associated with the frequency of contact exploration type (Figures 2 , 3a , b, and 4a ) and negatively with that of the short‐distance and the medium‐distance exploration types (Figure 3a,b ). Specifically, both root N and P concentrations were negatively associated with the frequency of short‐distance exploration type, while a quadratic relationship was found between the root P concentration and the frequency of the medium‐distance exploration type in a value range of 9%–30% (Figure 4b–d ). Clearly, variations in the length of emanates in A. faxoniana in response to varying environmental conditions was not explainable by adjustment in nutrient uptake capacity. This contradicts the findings of positive relationships between the nutrient status of host plants and the length of emanates of ECM roots in literature (Agerer, 2001 ; Brandes et al., 1998 ; Hobbie & Agerer, 2010 ; Lilleskov et al., 2011 ). A probable explanation for this contradiction is that, in natural ecosystems, while ECM symbionts respond to soil resource deficiency in the way of root proliferation and production of emanating hyphae, the consequence of improved nutrition in host plants could be offset by increased energy cost for the ECM root systems under multiple resource limitation. In this study, the soil N and P were mostly deficient across the study sites (alkaline N: 40.78 ± 18.83 mg/g, total P: 0.87 ± 0.26 mg/g). It is likely that the contact exploration type and the short‐distance exploration type mediated the uptake of alkaline N and available P, whereas the medium‐distance exploration type helped forage the organic N and P far from the root distal (Agerer, 2001 ; Hobbie & Agerer, 2010 ). Ostonen et al., ( 2007 ), Ostonen et al., ( 2011 ) noted that host trees can rely on the high efficiency of resource capture of the root–mycorrhiza continuum while investing little C to ECM root systems. Besides, plants would cut down the investment when C allocation outweigh the benefit obtained from ECM fungi (Johnson et al., 2003 ; Treseder, 2004 ), or ECM fungi sometimes hold the nutrients for themselves in priority while the host tree remains nutrient deficient under extreme nutrient limitations (Treseder & Allen, 2002 ). The improved root and foliar N and P by the occurrence of the contact exploration type and the negative relationships with the frequency of the short‐distance and the medium‐distance exploration types may partially attribute to trade‐offs between the C allocation to ECM emanates and nutrient uptake in host plants (Johnson et al., 2013 ; Magyar et al., 2007 ). Our findings allow us to develop a conceptual model on the intervention of ECM symbiosis on root and foliar N and P nutrition using A. faxoniana as a case study (Figure 5 ). The model illustrates that the ECM strategies strongly affect the root nutrients, and then through the interconnections between roots and aboveground tissues in nutrient transportation and re‐allocations, eventually influence the foliar nutrients, with preferential effects on P under both N and P limitations."
} | 4,304 |
39336255 | PMC11433202 | pmc | 6,217 | {
"abstract": "Polyvinyl alcohol (PVA), a versatile polymer, is extensively used across many industries, such as chemicals, food, healthcare, textiles, and packaging. However, research on applying PVA to triboelectric nanogenerators (TENGs) remains limited. Consequently, we chose PVA as the primary material to explore its contact electrification mechanisms at the molecular level, alongside materials like Polyethylene (PE), Polyvinylidene fluoride (PVDF), and Polytetrafluoroethylene (PTFE). Our findings show that PVA has the highest band gap, with the smallest band gap occurring between the HOMO of PVA and the LUMO of PTFE. During molecular contact, electron transfer primarily occurs in the outermost layers of the molecules, influenced by the functional groups of the polymers. The presence of fluorine atoms enhances the electron transfer between PVA and PTFE to maximum levels. Experimental validation confirmed that PVA and PTFE contact yields the highest triboelectric performance: V OC of 128 V, I SC of 2.83 µA, Q SC of 82 nC, and an output power of 384 µW. Moreover, P-TENG, made of PVA and PTFE, was successfully applied in self-powered smart devices and monitored human respiration and bodily movements effectively. These findings offer valuable insights into using PVA in triboelectric nanogenerator technologies.",
"conclusion": "4. Conclusions This study elucidates the CE mechanism between PVA and other polymers (PE, PVDF, PTFE) and identifies its potential for practical applications in the field of TENGs. By analyzing the energy gap between the HOMO and the LUMO, it can be demonstrated that PVA is more susceptible to electron transfer. It is worth noting that when PVA and PTFE molecules come into contact, the charge transfer primarily occurs within the outermost electron layer of the molecules. In the course of the performance experiments, the highest output performance was also achieved with a V OC of 128 V, an I SC of 2.83 µA, a Q SC of 82 nC, and a power output of 384 µW. The P-TENG was prepared by combining PVA and PTFE, and its capacity to power a thermometer was successfully verified. Furthermore, the P-TENG demonstrated the capacity to accurately monitor human respiratory activity, finger flexion, and movement status. This study not only advances our comprehension of the mechanism of action of PVA in the domain of friction nanogenerators, but also furnishes insights that can inform prospective innovations in self-powered and wearable electronic devices.",
"introduction": "1. Introduction Energy is a fundamental pillar in the advancement of human society. As science and technology progress, the scarcity of conventional energy sources escalates, worsening environmental contamination. In this era, dominated by the Internet of Things and sensor networks, the urgent demand for energy has highlighted TENGs as a key emerging energy conversion technology [ 1 ]. TENGs utilize the triboelectric effect, generating static electricity through friction to convert, often untapped, mechanical energy into electrical energy. This sustainable, clean, and self-powered method provides a promising solution for powering electronic devices [ 2 , 3 ]. Characterized by high output and energy conversion efficiency at low frequencies, TENG technology offers numerous advantages, including diverse material options, simple fabrication, low cost, with no need for external power, as well as being eco-friendly and sustainable. These benefits make TENGs a promising technology for applications in wearable electronics, flexible devices, self-powered sensors, and large-scale blue energy harvesting, driving sustainable energy development and utilization [ 4 , 5 , 6 ]. To date, the field of triboelectric charging has experienced rapid growth, covering aspects such as architectural design, material enhancement, performance optimization, power management, and exploratory applications [ 2 , 7 , 8 , 9 ]. Tribology, a complex process, involves contact or sliding between two materials. Various localized physical and chemical reactions occur during this interaction, such as deformation, heat generation, and surface layer formation [ 10 ]. The foundational, scientific phenomenon of TENG technology is contact electrification, wherein charge generation arises from the contact and separation of objects. Mechanical friction aids, but is not essential to, charge transfer. Almost all material types, including polymers, semiconductors, inorganic materials, and metals, can charge through contact electrification [ 11 , 12 ]. Polymeric materials, with their unique functional groups, mechanical properties, and physicochemical characteristics, are widely used in TENG technology [ 13 , 14 ]. In TENGs, electron-withdrawing groups include fluorine (-F), ester (-COOR), nitrile (-CN), carboxyl (-COOH), acyl (-CON), and nitro (-NO 2 ); electron-donating groups comprise amine (-NH 2 ), hydroxyl (-OH), amide (-CONH), and alkoxy (-OR) [ 15 , 16 ]. These functional groups enhance electron transfer and capture during the frictional contact–separation process. Moreover, polymeric films, notable for their flexibility, stretchability, processability, and lightweight nature, have been positioned as core materials in modern TENG technology. Polyvinyl alcohol (PVA), a semicrystalline polymer, is typically found in powdered form and synthesized through a two-step process. Synthesis begins with the free-radical polymerization of vinyl acetate into poly (vinyl acetate), which is then hydrolyzed to produce PVA [ 17 ]. Incomplete hydrolysis allows for PVA with varying degrees of hydrolysis, resulting in different solubility profiles and molecular weights. Consequently, PVA features a backbone of C-C macromolecular chains and numerous hydroxyl functional groups [ 18 ]. As a versatile molding material, PVA has been shaped into various forms, including films and coatings with high tensile strength, flexibility, and excellent odor barrier properties [ 19 , 20 ]. PVA is highly regarded as a triboelectric film due to its excellent hydrophilicity, good film-forming ability, mechanical flexibility, biocompatibility, chemical stability, tunable surface properties, and effective dielectric properties, making it ideal for efficient and sustainable energy harvesting applications. However, research on the role of PVA in triboelectric nanogenerators remains in the early stages [ 21 , 22 , 23 ]. In this study, we selected polyvinyl alcohol (PVA) as the primary material to systematically analyze its contact electrification mechanisms in response to various materials, its impact on the performance of a TENG, and certain practical applications. Initially, using Density Functional Theory (DFT), we investigated the contact electrification mechanisms between PVA and the representative polymers (PE, PVDF, and PTFE). The analysis covered the electrostatic potential, energy gap, and intermolecular charge transfer. The results show that PVA has a higher HOMO–LUMO gap, beneficial for electron transfer. When PVA contacts other polymers, electron transfer primarily occurs in the outermost electron layers, significantly influenced by the functional groups. The experimental results indicate that contact between PVA and PTFE yielded the highest electrical output. Applying these materials in a triboelectric nanogenerator (P-TENG) demonstrated their potential to power smart devices and monitor human respiration and bodily movements. This research lays a critical foundation for the application of PVA in triboelectric nanogenerators (TENGs)."
} | 1,883 |
28288560 | PMC5348893 | pmc | 6,218 | {
"abstract": "Background Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. Results We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of β-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. Conclusions MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0927-x) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions We found that intragenic concerted evolution within MiSp-encoding genes likely led to rapid proliferation of proline replacements for alanine or glutamine in MiSp protein sequences independently in at least two species. For one species, the proliferation of proline coincides with higher extensibility of minor ampullate silks. This could allow cobweb weavers to access new prey types or the ability to modulate mechanical properties of their silks through altering expression levels of MiSp gene copies. Our multiple nearly complete MiSp sequences also provide various templates for tailored biomimetic applications through recombinant DNA technologies.",
"discussion": "Discussion Our characterization of complete or almost complete encoding sequences for the minor ampullate spidroin (MiSp) in multiple cobweb weaving spider species (Theridiidae) demonstrate the presence of at least two MiSp loci in each species. The substantial variation of MiSp sequences within and among species and its relationship to variation in material properties has multiple implications for molecular evolution, spider ecology, and biomimetic applications through recombinant DNA technology. Two MiSp-encoding loci have also been documented in the golden orb-weaver spider, Nephila clavipes (Nephilidae), and therefore the presence of multiple MiSp-encoding loci in spider genomes most likely dates minimally back to the common ancestor of theridiids and nephilids (e.g. Araneoidea). Our reconciliation of gene trees with species trees suggests even older duplication events (Fig. 7 ). However, the maintenance of two loci appears to involve complex molecular evolutionary processes including intergenic concerted evolution of the loci within species, and multiple gains and losses of individual MiSp copies. Although our reconciliation analyses inferred independent duplication events within each of our cobweb weaving species, the grouping of MiSp loci within species based on the terminal domains (Figs. 6 and 7 ), could also result from intergenic concerted evolution of the N- and C-terminal encoding regions. We favor the latter hypothesis because of the dramatic differences in the repetitive region within some species (Fig. 3 ). Concerted evolution of the N and C-terminal encoding regions could occur through non-homologous recombination between the loci, facilitated by their similar sequences, as proposed for the major ampullate spidroin paralogs, MaSp1 and MaSp2 , of multiple species [ 2 , 32 , 41 – 43 ]. Within theridiids, the rate of concerted evolution appears to be faster than speciation, since relationships among each species’ pair of MiSp loci reflect species relationships (Figs. 6 and 7 ). Intergenic concerted evolution of terminal-encoding regions could be favored by selection because these regions are involved in assembly of multiple MiSp monomers into polymers and the conversion of the protein complex from a liquid to a solid [ 31 , 44 ]. Highly similar terminal regions may be necessary for polymers to form. Outside of theridiids, relationships among MiSp N and C-terminal domains are not congruent with species relationships. The low posterior probabilities and bootstrap support for these incongruous relationships suggest there is limited phylogenetic signal retained in the MiSp C-termini at the distant time scale of divergence of theridiids from other araneoid families (~170 million years ago, [ 36 , 38 , 39 ]). However, the consistent grouping of Nephila and theridiid MiSp C-terminal domains to the exclusion of araneid MiSp C-terminal domains suggests that nephilid and theridiid MiSp sequences are derived from a different ancient copy than the araneid MiSp sequences, as supported by our reconciliation analyses (Figs. 6 and 7 ). The loss of functional MiSp loci through multiple single nucleotide mutations is further supported by the presence of a MiSp pseudogene in the S. grossa genome. Many additional losses were inferred by our reconciliation analyses (Additional file 1 : Figure S8), but because of incomplete spidroin sampling for most species we do not feel confident in estimating the extent of spidroin gene loss. We found extensive variation in the MiSp repetitive regions between loci within a genome and among species (Fig. 3 ). MiSp variants in L. hesperus and L. tredecimguttatus, L. geometricus MiSp_v2 , P. tepidariorum MiSp_v1, Nephila MiSp 1 & 2, and araneid MiSp sequences are similar in terms of amino acid motif composition. S. grossa MiSp and L. geometricus MiSp_v1 are especially divergent, with the difference between variants within L. geometricus being extremely striking (Fig. 3 ). The divergent repeats have an increased proline content, which likely occurred independently in S. grossa MiSp and L. geometricus MiSp_v1, based on gene tree relationships (Figs. 6 & 7 ). The mutation of alanine or glutamine codons to a proline codon requires only a single base change and the GPG motif is frequently found as GPGA or GPGQ, indicating that it could have evolved from (GA) n or (GQ) n motifs, which are the most common motifs in all other MiSp sequences (Fig. 3 ). Intragenic concerted evolution could rapidly proliferate these mutations throughout a single gene as has been proposed for other spidroin paralogs [ 16 , 17 , 45 , 46 ]. The near identity of spacer sequences within each of our cobweb weaver MiSp variants (Fig. 4 ) supports the hypothesis that intragenic concerted evolution is common, as also found in orb-web weaver MiSp [ 30 , 31 ]. However, intragenic concerted evolution must be offset by some other molecular processes, since the remainder of the repetitive regions are not as homogenized as the spacers (Fig. 3 ). The simple amino acid motifs found in MiSp such as (GA) n are encoded by microsatellite-like sequences and could thus be prone to high rates of slipped strand mispairing. It is also possible that excessive homogenization of MiSp repetitive regions adversely affects its function and is eliminated by selection. The high proportion of the GPG amino acid motif in S. grossa MiSp correlates with higher extensibility in S. grossa minor ampullate silk fibers (Fig. 5c , Table 3 ). GPG content is probably not the only predictor of extensibility, however, since Latrodectus minor ampullate fibers were more extensible than orb-web weavers’ even though GPG content for L. hesperus is similar to the orb-web weavers. Furthermore, we did not find a significant difference in extensibility between L. hesperus and L. geometricus minor ampullate silk fibers despite L. geometricus MiSp_v1 having a relatively high percentage of GPG motifs (Fig. 3 ). The MiSp_v2 : MiSp_v1 transcript ratio in L. geometricus suggests that the GPG-rich MiSp_v1 is in lower abundance in L. geometricus (Table 2 ), which could explain why there is not a significant increase in extensibility of its minor ampullate silk fibers in comparison to L. hesperus . Cobweb weaving spiders use MiSp in their prey wrapping silks [ 5 ], and evolution of extensibility-conferring motifs could potentially allow for capturing different prey types by S. grossa compared to other cobweb weavers, although little is known about the ecology or diet of this species. It is also possible that L. geometricus modulates expression of its two MiSp-encoding loci in response to prey availability. Experimentally manipulating prey type has been shown to affect proline content of major ampullate fibers in Nephila , potentially as a result of plastic changes in relative expression levels of the proline-poor MaSp1 and the proline-rich MaSp2 [ 47 , 48 ]. Variation among individuals in MaSp1 and MaSp2 transcript abundance has also been demonstrated for black widows [ 49 ]. Analogous experiments have not been done for minor ampullate fibers. However, the variation in MiSp_v2 : MiSp_v1 ratios that we found between L. geometricus individuals (Table 2 ) suggests plasticity of expression is possible. Our six complete or nearly complete single exon MiSp -containing clones can also serve as templates for recombinant proteins. Due to the dramatic differences in length and amino acid content of some of the proteins encoded by these loci, it may be possible to spin artificial fibers with custom-made properties. For instance, the GPG-containing L. geometricus MiSp_v1 could be used to make more extensible fibers, while the longer alanine-rich L. hesperus MiSp_v1 may make stiffer fibers."
} | 2,689 |
39475655 | PMC11551391 | pmc | 6,219 | {
"abstract": "Significance Exploration into alternative plastic monomers to replace petrochemical-derived monomers has been limited due to production challenges. One promising class of potential alternatives is pseudoaromatic compounds. This study presents the sustainable production of five pseudoaromatic dicarboxylic acids using metabolically engineered Corynebacterium glutamicum strains directly from glucose. A base production strain was constructed to produce 2-pyrone-4,6-dicarboxylic acid, which was then further engineered and optimized to produce other pyridine dicarboxylic acids. Fed-batch fermentation of the engineered strains demonstrated significant production titers. This work provides insight into the metabolic flexibility and engineering potential for synthesizing bio-based aromatic monomers using C. glutamicum . The methodologies from this study will be useful for developing efficient microbial cell factories to produce various bio-based plastic monomers.",
"discussion": "Discussion In this paper, we developed five metabolically engineered C. glutamicum platform strains capable of producing pseudoaromatic dicarboxylic acids (PDC, 2,3-, 2,4-, 2,5-, and 2,6-PDCA) from glucose. A synthetic metabolic pathway toward PDC was constructed through the introduction of the C. testosteroni pmdABC operon ( 19 ). PDC production was optimized by the plasmid-based overexpression of the feedback-resistant aroG S180F gene, along with the asbF and ubiC genes, to increase the flux toward the precursor PCA. Genetic manipulation was performed to further increase the key precursor PCA supply by deleting the pcaHG, qsuD , and poxF genes, which are responsible for the competing pathways toward PCA biosynthesis, and by shifting the native glucose uptake system to use the PTS-independent glucose uptake system. This was achieved through promoter exchange of the iolT1 , iolT2 , and ppgk genes, and the deletion of the ptsH and iolR genes. Additionally, fed-batch fermentation conditions were optimized, resulting in the production of 62.03 g/L of PDC. Comparative transcriptome analysis led to the further overexpression of highly expressed genes (>50-fold) to assess their impact on PDC overproduction. Among 17 target genes, the PSE6 strain harboring plasmids pP3 and pP1-0160 produced 76.17 ± 1.24 g/L of PDC with an overall productivity of 0.63 ± 0.01 g/L/h and a yield of 0.36 ± 0.04 mol/mol glucose in fed-batch fermentation. The 2,3-, 2,4-, and 2,5-PDCA biosynthesis pathways were constructed, with the 2,3-PDCA route being a previously unreported biosynthetic pathway in this study. The two-step enzymatic conversions from their respective direct precursors were optimized in the high PDC-producing strain (PSE6) to achieve 2.79 ± 0.005 g/L of 2,3-PDCA, 494.26 ± 2.61 mg/L of 2,4-PDCA, and 1.42 ± 0.02 g/L of 2,5-PDCA in fed-batch fermentation, all of which were well above the product tolerance levels. To complete the portfolio of pseudoaromatic dicarboxylic acid production, L-lysine overproducing C. glutamicum BE strain and L-aspartate pathway–enhanced C. glutamicum BAL strains were engineered to produce 2,6-PDCA. Production of 2,6-PDCA in BAL10 strain was optimized by introducing four genes to increase the flux toward the precursor HTPA and by optimizing fed-batch fermentation conditions. The final engineered strain produced 15.01 ± 0.03 g/L of 2,6-PDCA in optimized fed-batch fermentation. This study marks a pivotal step in the sustainable production of pseudoaromatic dicarboxylic acids using engineered C. glutamicum , establishing a foundation and showcasing the potential of C. glutamicum as a microbial platform for producing plastic monomer alternatives. Furthermore, the metabolic engineering strategies described here offer a blueprint for designing microbial cell factories capable of efficiently producing chemicals of interest, including the pseudoaromatic chemicals described in this paper."
} | 984 |
34061185 | PMC8331144 | pmc | 6,220 | {
"abstract": "Abstract Mealybugs are insects that maintain intracellular bacterial symbionts to supplement their nutrient-poor plant sap diets. Some mealybugs have a single betaproteobacterial endosymbiont, a Candidatus Tremblaya species (hereafter Tremblaya ) that alone provides the insect with its required nutrients. Other mealybugs have two nutritional endosymbionts that together provision these same nutrients, where Tremblaya has gained a gammaproteobacterial partner that resides in its cytoplasm. Previous work had established that Pseudococcus longispinus mealybugs maintain not one but two species of gammaproteobacterial endosymbionts along with Tremblaya . Preliminary genomic analyses suggested that these two gammaproteobacterial endosymbionts have large genomes with features consistent with a relatively recent origin as insect endosymbionts, but the patterns of genomic complementarity between members of the symbiosis and their relative cellular locations were unknown. Here, using long-read sequencing and various types of microscopy, we show that the two gammaproteobacterial symbionts of P. longispinus are mixed together within Tremblaya cells, and that their genomes are somewhat reduced in size compared with their closest nonendosymbiotic relatives. Both gammaproteobacterial genomes contain thousands of pseudogenes, consistent with a relatively recent shift from a free-living to an endosymbiotic lifestyle. Biosynthetic pathways of key metabolites are partitioned in complex interdependent patterns among the two gammaproteobacterial genomes, the Tremblaya genome, and horizontally acquired bacterial genes that are encoded on the mealybug nuclear genome. Although these two gammaproteobacterial endosymbionts have been acquired recently in evolutionary time, they have already evolved codependencies with each other, Tremblaya , and their insect host.",
"introduction": "Introduction Insects with nutrient-poor diets (e.g., plant sap, blood, wood) maintain microbial symbionts that supplement their diet with compounds such as amino acids and vitamins ( Baumann 2005 ; Douglas 2006 ). Mealybugs ( fig. 1 A ) are insects that exclusively consume phloem sap and maintain nutritional endosymbiotic bacteria within specialized cells called bacteriocytes ( Buchner 1965 ; von Dohlen et al. 2001 ; Baumann et al. 2002 ). Mealybug bacteriocytes house between one and three different bacterial endosymbionts depending on the mealybug species ( Kono et al. 2008 ; Koga et al. 2013 ; López-Madrigal et al. 2013 ; Husník and McCutcheon 2016 ; Szabó et al. 2017 ; Gil et al. 2018 ). These mealybug endosymbionts produce essential amino acids and vitamins, which are present in plant sap at levels insufficient for insect growth. Although it is not uncommon for insects to simultaneously maintain multiple endosymbionts ( Buchner 1965 ; Fukatsu and Nikoh 1998 ; Thao et al. 2002 ; Toh et al. 2006 ; Moran et al. 2008 ; McCutcheon and Moran 2010 ), the spatial organization of the dual mealybug endosymbiosis is unusual: each bacteriocyte house cells of Candidatus Tremblaya princeps (betaproteobacteria, hereafter referred to as Tremblaya ), and inside each Tremblaya cell reside tens to hundreds of cells of another endosymbiont from the gammaproteobacterial family Enterobacteriaceae ( von Dohlen et al. 2001 ; Downie and Gullan 2005 ; Gatehouse et al. 2012 ), with the titer of gammaproteobacterial symbionts varying depending on host species and developmental stage ( Kono et al. 2008 ; Parkinson et al. 2017 ). Many of these intra- Tremblaya endosymbionts are members of the Sodalis genus, which are well-represented among endosymbionts of insects ( Toh et al. 2006 ; Clayton et al. 2012 ; Oakeson et al. 2014 ; Husník and McCutcheon 2016 ; McCutcheon et al. 2019 ; Hall et al. 2020 ). Fig. 1. The structure of the Pseudococcus longispinus symbiosis. ( A ) Image of P. longispinus mealybugs on a sprouted potato. ( B ) Montaged TEM overview image of a bacteriocyte from P. longispinus . The six to seven light gray blobs are Tremblaya cells, surrounding a central eukaryotic nucleus. Within each Tremblaya cell reside rod-shaped and more electron-dense gammaproteobacterial cells. Black-colored rods in between Tremblaya are mitochondria within eukaryotic cytoplasm. The insect nucleus is at the center of the bacteriocyte in a gray shade that is similar to Tremblaya . ( C ) Details from an electron tomographic slice showing the boundary of a Tremblaya cell, where a mitochondrion is visible near the Tremblaya cell envelope. ( D ) Higher magnification view of the mitochondrion shown in C . ( E ) Tomographic slice of a gammaproteobacterial symbiont that resides inside Tremblaya , showing numerous outer membrane vesicles (red arrows). The bacterial symbionts are easily distinguished from eukaryotic mitochondria. ( F ) Fluorescent in situ hybridization (FISH) image of P. longispinus bacteriome tissue showing the localization of two different gammaproteobacterial endosymbionts within Tremblaya cells. Fluorophore-labeled probes were used to localize Tremblaya cells (green) and the two gammaproteobacterial endosymbionts (yellow and magenta). Tremblaya cells that appear to harbor exclusively, or almost-exclusively, one type of gammaproteobacterial endosymbiont are circled in red. DNA and, therefore, insect nuclei were counterstained with Hoechst (white). Each nucleus is surrounded by several Tremblaya cells per bacteriocyte. ( G ) Zoomed in and annotated detail fluorescence microscopy image of a P. longispinus bacteriocyte. ( H ) Schematic representation of P. longispinus bacteriocytes. Genomic studies of numerous insect–endosymbiont systems have revealed strong and consistent patterns of complementary gene loss and retention among all members of the symbiosis ( Shigenobu et al. 2000 ; van Ham et al. 2003 ; Wu et al. 2006 ; McCutcheon and Moran 2010 ; Sloan and Moran 2012 ; Łukasik et al. 2018 ). Although, in most cases, a single endosymbiont genome will retain complete or near-complete pathways for individual metabolites, mealybug endosymbionts are unusual in that the reciprocal pattern of gene loss and retention exists within biochemical pathways ( McCutcheon and von Dohlen 2011 ; López-Madrigal et al. 2013 ; Husník and McCutcheon 2016 ; Szabó et al. 2017 ; Gil et al. 2018 ). Most of the previously published mealybug endosymbiont genomes were highly reduced in size (less than 1 Mb) and gene dense (containing few pseudogenes), which made discerning these complementary gene loss and retention patterns relatively straightforward. \n Pseudococcus longispinus harbors the symbiont Tremblaya , but unlike Tremblaya in other mealybugs, the P. longispinus strain of Tremblaya house not one but two gammaproteobacterial endosymbionts ( Rosenblueth et al. 2012 ; Husník and McCutcheon 2016 ). We previously reported draft genome assemblies of these two gammaproteobacterial endosymbionts, which suggested that their combined genome sizes were large, approximately 8.2 megabase pairs (Mbp) in length ( Husník and McCutcheon 2016 ). Phylogenetic analysis showed that one of these gammaproteobacterial symbionts belonged to the Sodalis genus, and the other was more closely related to members of the Pectobacterium genus. However, the poor quality of these draft genome assemblies made detailed genomic analysis impossible. Light microscopy on P. longispinus ( Gatehouse et al. 2012 ) suggested that the gammaproteobacterial endosymbionts reside inside Tremblaya cells, as is the case in other mealybugs ( von Dohlen et al. 2001 ). But it was unclear from these data whether one or both of these gammaproteobacteria are restricted to Tremblaya cells (i.e., if they are also in the cytoplasm of the host insect bacteriocyte), whether each gammaproteobacterial species is restricted to particular Tremblaya cell types, or whether the two gammaproteobacterial symbionts are mixed together inside undifferentiated Tremblaya cells. Here, we add long-read data generated from P. longispinus bacteriome tissue to greatly improve the gammaproteobacterial genome assemblies and annotations. We describe the relative cellular locations of the endosymbionts using fluorescence and transmission electron microscopy and report the genome evolutionary patterns and metabolic contributions of the microbial members of this unusual four-way symbiosis.",
"discussion": "Discussion Gammaproteobacterial Endosymbionts in P. longispinus Are of Recent Origin We conclude that the gammaproteobacterial endosymbionts in P. longispinus mealybugs have been introduced into a host-restricted lifestyle relatively recently, on a timescale roughly similar (tens to hundreds of thousands of years) to other young endosymbionts of insects and nematodes ( Toh et al. 2006 ; Burke and Moran 2011 ; Clayton et al. 2012 ; Boyd et al. 2016 ; Oakeson et al. 2014 ; Martinson et al. 2020 ). We base this conclusion on three features of their genomes. First, their genome sizes are large, comparable with those of free-living bacteria ( table 1 and fig. 3 ) ( Husník and McCutcheon 2016 ), showing that they have not yet undergone most of the genome reduction seen in more established bacterial endosymbionts ( McCutcheon and Moran 2011 ). Second, a phylogenomic tree, consisting of endosymbionts (old and young) and free-living bacteria, shows the gammaproteobacterial endosymbionts of P. longispinus on short branch lengths ( fig. 2 ), indicating that they have not yet experienced the rapid sequence evolution typical of older endosymbiotic bacteria ( Moran 1996 ). The branch lengths of P. longispinus gammaproteobacteria are similar to those of other recently acquired endosymbionts and are substantially shorter than the branch lengths of older symbionts that have undergone millions of years of genome erosion. Third, their GC-contents at 4-fold degenerate sites in coding regions remain relatively high (supplementary file 4, Supplementary Material online), whereas older endosymbionts typically show pronounced AT biases at these sites ( Wernegreen 2002 ; Van Leuven and McCutcheon 2012 ). We attempted to infer which gammaproteobacterial endosymbiont might have been established first within P. longispinus bacteriocytes. The lower average d S and shorter branch length relative to its closest free-living relative ( fig. 2 ) suggests that Sod. endolongispinus is the younger of the two gammaproteobacterial endosymbionts in P. longispinus . This is consistent with our rough estimate of 68,000 years as the divergence time between Sod. endolongispinus and Sodalis HS compared with an estimated divergence of 466,000 years for Sym. endolongispinus and P. carotovorum ( Martinson et al. 2020 ). It is important to emphasize that these dates are extremely speculative. Additionally, it is possible that the longer branch length of Sym. endolongispinus (as well as the other Symbiopectobacterium symbionts) relative to the free-living Pectobacterium / Brenneria spp. is due to the fact that a close relative to the Symbiopectobacterium has not yet been sequenced. Creation of Pseudogenes Is Likely Coupled with Rapid Deletion During their brief period of host restriction and vertical transmission, Sym. endolongispinus and Sod. endolongispinus have accumulated thousands of pseudogenes ( fig. 3 ). This is in stark contrast to nonendosymbiotic bacteria, where pseudogenes have been reported to account for only 1–5% (in some cases, as high as 8%) of the genetic repertoire ( Liu et al. 2004 ; Lerat and Ochman 2005 ). The high level of gene inactivation in both Sym. endolongispinus and Sod. endolongispinus genomes is caused by many frameshift-causing indels and nonsense mutations, resulting in either truncated, run-on, and fragmented genes (supplemental files 3 and 4, Supplementary Material online). A small proportion of apparently functional genes appear to be cryptic pseudogenes, or genes that have elevated d N /d S values (>0.3) relative to orthologs in a closely related nonendosymbiont genome ( Clayton et al. 2012 ; Oakeson et al. 2014 ; Van Leuven et al. 2014 ; Burke and Moran 2011 ). However, the vast majority of both intact and broken genes have low d N /d S values consistent with strong purifying selection. This suggests that pseudogenes in Sym. endolongispinus and Sod. endolongispinus have formed very recently and have not yet had time to accumulate substitutions that would elevate their d N /d S values. As noted in previous studies of pseudogene flux in several strains of Salmonella ( Kuo and Ochman 2010 ) and consistent with the previously reported deletional bias in bacterial genomes ( Mira et al. 2001 ; Kuo and Ochman 2009 ; Burke and Moran 2011 ), it is likely that deletion of pseudogenes happens quickly, on time scales shorter than these genes can accumulate significant numbers of nonsynonymous sequence substitutions. Recent Transposase Expansion a Common, but Not Universal, Feature of Early Genomic Disruption in Endosymbionts \n Sod. endolongispinus encodes 220 transposases, over an order of magnitude greater than its closest sequenced free-living relative. The high number of transposases in Sod. endolongispinus is consistent with previous reports of IS expansion as a by-product of relaxed selection on large parts of the genome as well as a mechanism for genome rearrangement and reduction ( Mahillon and Chandler 1998 ; Plague et al. 2008 ; Belda et al. 2010 ; Schmitz-Esser et al. 2011 ; Oakeson et al. 2014 ; Hendry et al. 2018 ). Indeed, other recently acquired Sodalis endosymbionts (SOPE and S. glossinidius ) also encode high numbers of transposases ( Clayton et al. 2012 ; Oakeson et al. 2014 ). Each Sodalis endosymbiont encodes different types and distributions of IS families (supplementary fig. 2 A , Supplementary Material online). Certain IS families present in the other Sodalis endosymbionts encode transposases that are not found in Sodalis HS (e.g., IS21 in SOPE, IS1 in Sodalis sp. SCIS). Given that ISs are highly dynamic, moving within and between genomes ( Touchon and Rocha 2007 ), it is possible that even a very close relative to Sodalis HS could have radically different types and distributions of ISs. Similar to Sod. endolongispinus , SOPE and S. glossinidius have likely undergone an IS expansion very recently, since the vast majority of the transposase sequences fall into only a handful of sequence clusters composed of nearly identical copies of just a few families per genome (supplementary file 1 and supplementary fig. 2 B , Supplementary Material online) ( Gil et al. 2008 ; Oakeson et al. 2014 ). Because transposases are known for facilitating genomic deletions ( Mahillon and Chandler 1998 ) and have proposed to be involved in genome reduction in endosymbionts ( Siguier et al. 2014 ), it is possible that this deletional tendency of transposases results in the elimination of IS elements themselves over time ( Plague et al. 2008 ; Schmitz-Esser et al. 2011 ; Siguier et al. 2014 ). Consistent with this idea, the genomes of older endosymbionts encode no or very low numbers of IS elements (supplementary fig. 2, Supplementary Material online). IS proliferation does not appear to be a universal phenomenon in young endosymbionts, at least not among the Sodalis and Symbiopectobacterium endosymbionts screened here. Several Sodalis endosymbionts (e.g., Sodalis sp. TME1, Sodalis sp. SCIS), whose genomes are comparable in size to Sod. endolongispinus (supplementary table 1, Supplementary Material online), appear to have few or no transposases (supplementary fig. 2, Supplementary Material online). None of the Symbiopectobacterium symbionts encodes nearly as many transposases as Sod. endolongispinus or S. glossinidius but has similar or lower numbers of transposases relative to P. wasabiae and P. cartovorum , the closest nonendosymbiotic relatives of the Symbiopectobacterium clade. The relative lack of transposases is surprising, given the vast amount of superfluous genome space in these young endosymbiont genomes in which transposases could insert themselves (supplementary table 1, Supplementary Material online). It is possible that there are large and diverse populations of free-living Sodalis and Symbiopectobacterium strains in nature that vary in IS content, and that the amount of IS proliferation that occurs in a newly established endosymbiont reflects the IS load of the ancestral free-living strain. Although it is also possible that the dearth of detectable transposase genes is caused by the low assembly quality of some of the endosymbiont genomes that are highly fragmented (e.g., >100 contigs) (supplementary table 2, Supplementary Material online). The highly fragmented nature of these short-read assemblies can, in part, be caused by the prevalence of identical or nearly identical transposases that cannot be resolved using short reads alone. For example, the assembly corresponding to Sym. endolongispinus published by Martinson et al. (2020) comprised 83 contigs (using only the Illumina reads published by Husník and McCutcheon 2016 ), with only 6 transposases detected using the ISfinder software that is included in the Prokka annotation pipeline. Our hybrid assembly using PacBio and Illumina reads resolved this genome into 9 contigs, from which we were able to identify 36 transposases. Similarly, our Illumina-only assembly of the Sod. endolongispinus genome resulted in 109 contigs, with only 7 identifiable transposases; our PacBio/Illumina hybrid assembly resolved the genome of Sod. endolongispinus into 3 contigs with 220 identifiable transposases. It seems likely that the estimated number of transposases in genomes generated from short-read data alone are significantly underestimated, with near-identical ISs collapsing into small unassembled contigs. Establishment of Interdependence Occurs Early During Endosymbiont Establishment Previous research has demonstrated that newly evolved endosymbiotic bacteria lose genes in response to the preexisting genetic inventory of their cosymbionts and (if present) HGTs on the host genome ( Wu et al. 2006 ; McCutcheon and Moran 2007 ; Nikoh and Nakabachi 2009 ; McCutcheon et al. 2009 ; Sloan and Moran 2012 ; Husník et al. 2013 ; Luan et al. 2015 ; Nowack et al. 2016 ). The occurrence of two recently acquired endosymbionts in P. longispinus presented us with a unique opportunity to investigate the inception of genomic complementarity and metabolic interdependence in a complex four-way symbiosis. During this early period of host restriction, we might expect to see more rapid gene loss in pathways whose precursors, intermediates, and products are more easily transported between different members of the symbiosis. This can include metabolites for which dedicated transporters (e.g., amino acid permeases) already exist in the free-living predecessor. For example, Sod. endolongispinus encodes genes for the transport of histidine and biotin, possibly contributing to the rapid loss of the biotin and histidine biosynthesis pathways in that endosymbiont ( fig. 4 ). Many genes that are part of amino acid and vitamin metabolism are either encoded on the host’s nuclear genome as HGTs from bacteria or on Tremblaya ’s diminutive genome, relieving the need for these new gammaproteobacterial symbionts to continue maintaining these genes. Consistent with this, we observe loss and pseudogenization of many pathway components for amino acid and vitamin biosynthesis in Sym. endolongispinus and Sod. endolongispinus ( fig. 4 A ), suggesting that genes in these pathways are lost rapidly and in response to genes already present in the symbiosis. As Sym. endolongispinus and Sod. endolongispinus are relatively new to a host-dependent lifestyle, they still encode many genes redundant with other genes present in the system ( fig. 4 B ). Most of these redundant genes appear to be undergoing strong purifying selection, evident from their low d N /d S values (supplementary files 3 and 4, Supplementary Material online). Because other older and longer-established mealybug symbioses show little evidence of genetic redundancy across genomes ( Husník and McCutcheon 2016 ), we suspect that many of the redundant genes in the gammaproteobacterial endosymbionts simply have not had a chance to accumulate substitutions that would break genes, elevate their d N /d S values, or delete them completely from one genome or the other. Loss of Core Metabolic and Structural Genes Occurs More Slowly In contrast to the rapid gene loss in pathways for amino acid and vitamin biosynthesis, genes in pathways for peptidoglycan biosynthesis and central metabolism are more strongly conserved in both of the gammaproteobacterial symbionts of P. longispinus . We have previously demonstrated that in Moranella , the ancient gammaproteobacterial symbiont of P. citri , peptidoglycan biosynthesis occurs in concert with bacterial genes encoded on its host’s nuclear genome as HGTs ( Bublitz et al. 2019 ). Consequently, Moranella has lost much of its peptidoglycan biosynthesis pathway and is presumably reliant on the import of host-derived proteins. Although this level of cellular integration represents a potential future state for Sym. endolongispinus and Sod. endolongispinus , it appears that this level of integration has not yet been achieved in the relatively short amount of time that these symbionts have been inhabiting P. longispinus . We hypothesize that the generally stronger retention of the peptidoglycan biosynthesis pathway throughout the early stages of endosymbiosis is due to the difficulty of integrating pathways that require the shuttling of complex molecules (such as PG precursors) or proteins between different cellular compartments. The loss of a key gene of the peptidoglycan biosynthesis pathway ( murF ) in Sod. endolongispinus is therefore quite interesting given that the protein product of this gene has been shown to be imported by the gammaproteobacterial endosymbiont in a related mealybug ( Bublitz et al. 2019 ). It is possible that the repeated recruitment and maintenance of endosymbionts from the Sodalis genus ( Husník and McCutcheon 2016 ) has made mealybugs particularly suited for rapid cellular integration of Sodalis relatives after infection. Rapid Sodalis adaptation to mealybug endosymbiosis is consistent with stronger conservation of peptidoglycan biosynthesis in Sym. endolongispinus , even though we estimate that Sym. endolongispinus is the older of the two gammaproteobacterial endosymbionts. More rapid Sodalis adaptation is also supported by the patterns observed in the degradation of pathways for the synthesis of essential amino acids and vitamins: out of those pathway components that are encoded by Tremblaya or the host genome as HGTs, 21 genes are lost by Sod. endolongispinus , whereas only 11 are lost by Sym. endolongispinus ( fig. 4 ). Members of the Symbiopectobacterium clade do not seem to commonly infect mealybugs, as only one of seven mealybug species for which we have genomic data houses a Symbiopectobacterium -related symbiont ( Husník and McCutcheon 2016 ; Szabó et al. 2017 ). A possible preference of mealybugs toward recruitment of Sodalis -related endosymbionts may be due to a combination of factors, including as of yet unknown factors within the host, as well as the genetic repertoire of the infecting bacteria, although it just may be that mealybugs interact more frequently with Sodalis in nature."
} | 5,931 |
25346794 | PMC4203501 | pmc | 6,221 | {
"abstract": "A successful honey bee forager tells her nestmates the location of good nectar and pollen with the waggle dance, a symbolic language that communicates a distance and direction. Because bees are adept at scouting out profitable forage and are very sensitive to energetic reward, we can use the distance that bees communicate via waggle dances as a proxy for forage availability, where the further the bees fly, the less forage can be found locally. Previously we demonstrated that bees fly furthest in the summer compared with spring or autumn to bring back forage that is not necessarily of better quality. Here we show that August is also the month when significantly more foragers return with empty crops ( P = 7.63e-06). This provides additional support that summer may represent a seasonal foraging challenge for honey bees."
} | 207 |
36819875 | PMC9930989 | pmc | 6,222 | {
"abstract": "One restriction for biohybrid photovoltaics is the limited conversion of green light by most natural photoactive components. The present study aims to fill the green gap of photosystem I (PSI) with covalently linked fluorophores, ATTO 590 and ATTO 532. Photobiocathodes are prepared by combining a 20 μm thick 3D indium tin oxide (ITO) structure with these constructs to enhance the photocurrent density compared to setups based on native PSI. To this end, two electron transfer mechanisms, with and without a mediator, are studied to evaluate differences in the behavior of the constructs. Wavelength-dependent measurements confirm the influence of the additional fluorophores on the photocurrent. The performance is significantly increased for all modifications compared to native PSI when cytochrome c is present as a redox-mediator. The photocurrent almost doubles from −32.5 to up to −60.9 μA cm −2 . For mediator-less photobiocathodes, interestingly, drastic differences appear between the constructs made with various dyes. While the turnover frequency (TOF) is doubled to 10 e − /PSI/s for PSI-ATTO590 on the 3D ITO compared to the reference specimen, the photocurrents are slightly smaller since the PSI-ATTO590 coverage is low. In contrast, the PSI-ATTO532 construct performs exceptionally well. The TOF increases to 31 e − /PSI/s, and a photocurrent of −47.0 μA cm −2 is obtained. This current is a factor of 6 better than the reference made with native PSI in direct electron transfer mode and sets a new record for mediator-free photobioelectrodes combining 3D electrode structures and light-converting biocomponents.",
"conclusion": "4. Conclusions In the study at hand, photobiocathodes based on 3D ITO are described, which exploit fluorophore-modified PSI to close its green gap. The dyes can be proven to provide an additional signal at their absorbance maxima in wavelength-dependent measurements and contribute significantly to the constructs' performance. In one approach of electrode construction, cyt c has been applied as a mediator to shuttle electrons to PSI. Here, all modified photosystems outperform the natural one up to a factor of about two. The TOF has also been increased for all constructs; up to 105 e − /PSI/s have been measured for photosystems that have been altered with both dyes, ATTO 590 and ATTO 532. Significant differences are obtained for the mediator-less approach (DET mode). Since PSI-ATTO590 weakly binds to the ITO structure, its photocurrent is lower than that of the reference specimens, although the TOF is doubled. In contrast, PSI-ATTO532 binds nearly as effectively as native PSI to the 3D ITO and the dye most likely aids in the orientation of the construct towards the surface. Thus, the photocurrent density and TOF increased tremendously to −47.0 μA cm −2 and 31 e − /PSI/s, respectively. The findings presented, here, offer many new opportunities, especially for mediator-less approaches, since major differences have been obtained depending on the modification. Another interesting prospect of biohybrid photovoltaics is the coupling of the photoactive setup to enzymes for the conversion of specific molecules. Therefore, high photocurrents are desirable because they provide more electrons. However, efficient communication between all components is essential as well. By now, it remains open whether this interaction is impeded or aided by the presence of the fluorophores. Due to this study, the stimulating question emerges what other modifications might be advantageous for PSI or other biocomponents in general for the interaction with artificial materials. In the present work, only minor changes have been made (by coupling 11 to 19 fluorophores to the large trimeric PSI complex), leading to massive behavioral alterations. The performance of PSI-ATTO532 on 3D ITO in DET mode is boosted for all wavelengths; hence, this aspect is not limited to light-converting components. Since the interaction is generally improved, the findings presented, here, will most likely lead to advances for various photobioelectrodes or bioelectrodes based on DET.",
"introduction": "1. Introduction Earth's main power source is solar radiation. For over 3 billion years, nature has converted this resource in a process called photosynthesis to produce energy-rich carbohydrates and molecular oxygen as a byproduct. 1–4 In plants and cyanobacteria, this procedure relies on two photoactive protein complexes, photosystems I and II (PSI and PSII). While PSII provides electrons by water-splitting, PSI leads to the reduction of NADP + in a multi-step process, which is afterward utilized for CO 2 fixation. 2,5–7 In recent years, many efforts have been made to use this natural potential for the benefit of humanity or, more specifically, for sunlight to electricity conversion. 8–10 In biohybrid photovoltaics, photoactive biocomponents are connected to artificial electrode materials to generate that photocurrent. Successful setups have been achieved with PSI, 1,5,7,11–15 PSII, 9,16–21 and bacterial reaction centers. 4,8,22–25 But likewise, thylakoid membranes 26–28 and even whole cells 29–32 have been deployed effectively. Many electrode materials and constructions have been applied as the synthetic part in contact with the biological entity. Metals, alloys, and metal oxides such as gold, 8,11,24,33,34 silver, 22 indium bismuth tin, 31 antimony tin oxide (ATO), 25,35 indium tin oxide (ITO), 14,21,28,36–38 titanium dioxide, 16,20,39,40 zinc oxide, 8,29 and zirconium dioxide 39 as well as many carbon-based materials 1,11,21,41,42 have been utilized successfully. It is important to note that not only flat 2D electrodes have been produced, 24,34,43 but also thicker architectures including multilayers, 13,14,24,33 hydrogels, 1,7,23 and 3D setups. 18,20,38,41,44 This is mainly devoted to better light energy usage and reflects the photoactive components' arrangement in a natural thylakoid membrane. A critical aspect of the overall performance of such biohybrid systems is the interaction of the biocomponent with the electrode material. As a defined orientation of large photosystems is difficult to achieve, often redox-active molecules have been used to shuttle electrons. Examples for these mediators are small redox molecules, 5,18,26,30,32,42 redox polymers 17,19,23,27,28,32,34 or different cytochromes. 12,22,27,35,36,45 The renunciation of redox mediators and, thus, the exploitation of a direct electron transfer provide a less complex system with better-defined electron transfer pathways. However, in general, these approaches result in significantly lower photocurrent densities. 18,32,41,44 Uncountable steps of evolution have optimized photosynthesis. Once a photosystem captures a photon, it is converted with almost 100% internal quantum efficiency. 2,5,7,14,46 For sunlight absorption, both complexes, PSI and PSII, mainly rely on the green pigment chlorophyll. 1,8,10 Hence, they cannot convert green light efficiently. This limited absorption in the region between 500 and 650 nm is termed the green gap and coincides with the area where the sunlight reaches its peak. 4 Thus, this property of photosystems is one of the main causes for their low external quantum efficiency. 1,2,4,47 Both nature and science have tried to reduce this limitation. Photosystems do not solely rely on photon absorption by chlorophylls but also contain other pigments. While one primary purpose of carotenoids is the prevention of oxygenic stress, they are also excited by light in the green gap. 48,49 This absorbed energy can be transferred to the chlorophylls, thus increasing the performance of the photosystems. 49,50 Much more efficient, however, are the phycobilisomes in cyanobacteria and red algae. These light-harvesting antenna complexes surround the reaction centers and work well in their absorption gap. 8,51 In research, photoactive biocomponents have been combined with many different systems that can interact with green light. Here, two different principles can be distinguished. On the one hand, a 2 nd photoactive component is integrated into the electrode structure. Here, charge separation occurs in the biocomponent as well as in the 2 nd component. Hence, higher cell voltages are obtained and the photoelectrochemical output is improved. 16,20,40 On the other hand, additional light-harvesting dyes have been coupled to photosystems following the natural example to increase the number of excited electrons. It can be shown that Förster resonance energy transfer (FRET) works for covalently connected synthetic systems, e.g. , from Rhodamine Red to the light-harvesting complex, 52 CdTe quantum dots to purple bacterial reaction centers, 53 and Lumogen Red to PSI. 54 Dutta et al. have attached three types of Alexa Fluor dyes to genetically modified bacterial reaction centers. Thus they obtained more than twice as much charge separation as in unmodified reaction centers. 55 Gordiichuk et al. covalently modified PSI with artificial ATTO 590 dyes and thus improved the electron transfer to oxygen up to 4-fold. 56 However, despite the many studies demonstrating the upgrading of light-harvesting protein complexes with artificial entities, only a few approaches have been described for applications in photobioelectrodes. Yoneda et al. attached fluorophore-modified reaction centers to ITO electrodes and proved a significant photocurrent linked to the absorption maxima of the dyes. 36 This group also demonstrated that the efficiency of excitation energy transfer increases with raising spectral overlap. 57 Yet, overall the photocurrents were very low for both systems. 36,57 Hartmann et al. modified PSII with phycobilisomes of three different cyanobacteria and thereby doubled the incident photon-to-current conversion efficiencies in the green gap. 17 Yet, so far, the overall performance of a biohybrid system has not been improved by any modification with synthetic dyes. The aim of the present study is the first construction of a photobioelectrode based on PSI, which was modified with covalently bound fluorophores. ATTO 590 has been chosen as an artificial dye since the FRET to PSI was proven to be very effective. 56 A three-dimensional structure of ITO nanoparticles has been selected as the synthetic electrode material because of its advantageous properties, particularly a high surface area and good transparency. 15,37,44 Regarding the interaction of the artificial electrode surface with PSI, a mediator based approach exploiting the small redox protein cyt c (MET) 37 has been utilized. Additionally, the direct electron exchange of native PSI and the modified PSI constructs with the ITO surface (DET) has also been investigated – based on our recent study. 58 To achieve more than solely a proof-of-principle, this study aims to elucidate the potential of PSI modification for a significant increase in photocurrent density. Thus, another fluorophore (ATTO 532) with a different excitation wavelength has been included in the investigations. Furthermore, PSI constructs with both fluorophores, ATTO 532 and ATTO 590, are evaluated. The study will emphasize that not only conditions of efficient FRET are necessary when modified photoactive complexes are combined with electrodes, but also a productive interaction of the artificial material and the modified biocomponents.",
"discussion": "3. Results and discussion 3.1 Characterization of the PSI-ATTO-constructs The present work aims to increase the photocurrent density of photobioelectrodes by modifying PSI with synthetic fluorophores. Here, two dyes (ATTO 590 and ATTO 532) have been selected with two different absorption maxima in the green gap ( ca. 590 nm and 532 nm respectively; for absorbance spectra see Fig. S2 † ). These molecules have been covalently coupled to PSI via NHS chemistry (see Section 2.2). Additionally, to the single fluorophore coupling to PSI, both fluorophores have also been bound. The latter construct is termed, here, PSI-ATTO-mix. As a first step, the constructs have been examined to verify the successful coupling. To this end, UV/vis spectroscopy has been conducted in buffer D solution. All three constructs have been purified by dialysis after the coupling reaction. In the solution, there is no unbound fluorophore present since no fluorophore could be detected in the dialysate during purification. Therefore, the signals at 590 and 532 nm, respectively, can be attributed to covalently attached dyes. Fig. 1A shows clearly that the coupling has been successful. Distinct peaks are visible in the green gap, where PSI does not absorb well as shown by the reference spectrum. The spectra can also be used to quantify the coupling efficiency. A summary of the obtained coupling ratios is given in Table 1 . For comparison, only data of the coupling of ATTO 590 can be found in the literature, and the results are similar. 56 Slight differences occur since for the present investigations high PSI concentrations are necessary for the subsequent immobilization in the 3D electrode structure. Thus, for all coupling experiments, a PSI concentration of 28 μM (compared to ca. 1 μM in the literature) has been used. Fig. 1 (A) to (C) Spectra of the constructs and pure ATTO dyes in buffer D. (A) Absorbance of the PSI-ATTO variants normalized at 680 nm; (B) fluorescence spectra after excitation at 532 nm (ATTO532_pure: ATTO dye in absence of PSI); (C) fluorescence after excitation at 590 nm (ATTO590_pure: ATTO dye in absence of PSI); (D) comparison of the oxygen consumption of the different PSI constructs in solution with cyt c as the electron donor (see Section 2.6). Pure: unmodified PSI ( n = 3); ATTO590: PSI-ATTO590 ( n = 3); ATTO532: PSI-ATTO532 ( n = 4); ATTO-mix: PSI-ATTO-mix ( n = 3). Degree of labeling (DoL) for the 3 modified PSI used in this study. The values were determined by UV/vis spectroscopy in buffer D and are stated as dyes per PSI trimer (n.a. – not applicable) Construct DoL (ATTO 590) DoL (ATTO 532) PSI-ATTO590 11 n.a. PSI-ATTO532 n.a. 12 PSI-ATTO-mix 8 11 To evaluate the FRET within the constructs, fluorescence measurements have been executed. Fig. 1B and C show that the fluorescence of all the tested chromophores is drastically quenched when they are connected to PSI compared to pure ATTO molecules in solution. This indicates that FRET is feasible for the three constructs. For the PSI-ATTO532 variant, a small fluorescence peak remains while it is not present for PSI-ATTO590. The latter is in line with the literature. 56 This implies that FRET in the PSI-ATTO590 construct is slightly more effective due to the higher overlap of the fluorescence emission with the absorption of PSI. Yet, for the PSI-ATTO532 construct also a significant energy transfer is achieved as the fluorescence peak's height is reduced by one order of magnitude. In the PSI-ATTO-mix construct, only a small fluorescence signal remains after excitation. This indicates that here energy transfer is slightly more efficient than with only ATTO532 coupling to PSI. The FRET can also be verified by evaluating the PSI fluorescence with excitation at 532 nm or 590 nm (see Fig. S3 † ). Here the fluorescence intensity is increased after coupling the dyes to the photoactive complex. Furthermore, the activity of the different constructs has been assessed. Thereto, the O 2 consumption has been determined under illumination with cyt c as the electron donor. The results shown in Fig. 1D illustrate that all variants are very active. The modified PSI constructs perform significantly better since they can absorb more photons from green light due to the attached dyes. The O 2 consumption is approximately doubled for all modifications. There is no clear trend visible. As indicated in the fluorescence measurements, the performance of the PSI-ATTO532 variant is also boosted despite the lower spectral overlap. After demonstrating successful coupling and functional energy transfer for all three PSI constructs, they have been subsequently studied as a photoactive component in photobiocathodes. 3.2 Working principle The properties of the photoactive protein complex have been altered by attaching the dyes to the trimeric PSI from Thermosynechococcus vesticus as indicated in Fig. 2 . Such coupling affects not only the absorption spectrum, but also other properties. For example, the modified PSI is no longer soluble in 5 mM MES pH 6 with added 60 mM MgSO 4 and 0.02% DDM, although this buffer works well for native PSI. 60 Hence, its behavior in photobioelectrodes might also be impacted and will, thus, be investigated here. Fig. 2 Working principle of the photobiocathode. Top left: Sketch of the preparation of the artificial 3D ITO structure by spin-coating and a baking step. Top right: Coupling PSI and the 2 ATTO dyes and illustration of their different UV/vis absorbance properties. Symbolization of the constructs: PSI-pure (a), PSI-ATTO590 (b), PSI-ATTO532 (c), and PSI-ATTO-mix (d). Center: Photographs of biohybrid setups prepared by immobilization of the constructs onto 3D ITO (labeling (a) to (d) as before). Bottom: Electron pathways for the two evaluated electron transfer mechanisms. When light hits the electrode, only non-green light can be converted by PSI. ATTO 590 and ATTO 532 are excited by light in this green gap. Curved arrows indicate the direction of the energy flow via FRET. Two principles are exploited to refill the electron into P700, MET via cyt c (left) or DET without a mediator and direct interaction of the ITO with the luminal side of the protein complex (right). For the study, 3D ITO electrodes have been used, which can be prepared easily by a template-based approach combining spin-coating depositions with a heating step for template removal and ITO sintering. This procedure allows tuning the thickness in the range of 10 to 30 μm providing thus an attractive 3D space for PSI immobilization. 58 The open 3D structures have been verified by SEM investigations as given in Fig. S4. † Two strategies have been tested to connect PSI and the three PSI constructs with the electrode: (i) a mediator-based approach applying cyt c and (ii) a mediator-free setup with direct electron exchange between ITO and PSI. The former has already been observed several times in the literature. 15,37,38 For the latter, it has been shown recently by ourselves that conditions can be found to increase the performance significantly. 58 For the immobilization of the native protein and the constructs, direct assembly on 3D ITO has been exploited which is fast and efficient. The same is valid for cyt c deposition. This redox active protein was co-immobilized with PSI in some of the photobiocathodes to shuttle the electrons to PSI. The mediation of electron transfer between PSI and electrodes by means of the small redox protein has been previously carefully analyzed and is thus not in the focus of the present study. 34,37,60 The following conditions have been applied for all constructs – 4 μM PSI solutions for electrode preparation in DET mode and 20 μM PSI and 1 mM cyt c solutions for MET for 3 min each. However, the different surface properties of the different PSI variants may influence the adsorption efficiency within the short time interval of immobilization, as studied in Section 3.6. In all the experiments, no additional electron acceptor has been added to the solution so that the oxygen in the air-saturated buffer is the final electron acceptor. 35,62 In the case of cyt c containing electrodes (MET), the electrons are transferred from the electrode towards this redox protein first and then to the excited PSI. The electrons are transported along the in-built electron transfer chain towards the stromal side where they can be passed to molecular oxygen. For cathodes without cyt c and, thus, without any mediator present in the setup, the electrons are transferred directly from the ITO material to the luminal side of the PSI complex (DET). Then the electrons follow the same pathway as described for the MET system. Different photoelectrochemical techniques have been applied to characterize the electrodes with PSI and the three PSI constructs, as will be explained in the following three subsections. They confirm the behavior of PSI-ITO hybrid structures as photobiocathodes. Finally, it must be noted that for ITO/PSI/cyt c electrodes, a buffer E (5 mM PPB pH 7) is used during operation, whereas for the ITO/PSI electrodes in DET mode, buffer F (100 mM MES buffer pH 6, and 400 mM KCl) is applied. These conditions provide a suitable setting for the respective setups. 37,58 3.3 Potential behavior of the photocurrent The first series of tests have been performed with chopped-light voltammetry on electrodes with the different constructs. Therefore, the applied potential has been varied in a wide range while white light at 100 mW cm −2 has been turned on and off repeatedly. The results of such experiments are compiled in Fig. 3 . At first glance, it is evident that the setup of photobioelectrodes is successful for all constructs for both MET and DET as envisioned in Section 3.2. While only slight signs of anodic currents have been obtained at higher applied potentials, clear photocathodic responses can be verified. Fig. 3 Chopped-light voltammetry and determination of the onset potential of the cathodic photocurrent and the optimal working potential for the photobioelectrodes based on (A and B) MET and (C and D) DET. Yellow bars in A & C are exemplary for one 5s-window, where the light is turned on. For better visibility some curves are shifted in D with +4 μA cm −2 (PSI-pure), +2 μA cm −2 (PSI-ATTO590), and −2 μA cm −2 (PSI-ATTO-mix). The experiments also enable the determination of the onset potential of the cathodic photocurrent. The electrodes based on mediation depend on the redox potential of the applied cyt c . Here, cathodic photocurrents start at about +100 mV vs. Ag/AgCl for all constructs. This finding is in line with the literature. 37 By decreasing the potential, the photocurrent can be further enhanced, but potentials lower than −100 mV vs. Ag/AgCl do not increase the current output substantially. For DET, the first cathodic photocurrents are obtained at higher potentials. Interestingly, here, significant differences appear for the constructs. The highest onset potential can be found for the unmodified PSI. At 400 mV vs. Ag/AgCl, it corresponds well to the values seen before. 58 The cathodic photocurrents for the constructs start at slightly lower potentials ranging from 250 to 325 mV vs. Ag/AgCl. Also, for the electrodes operating in DET mode, a lower potential has been found beneficial for a further gain in photocurrent. From these evaluations, the working potential for the photocurrent measurements can be determined as well. For all experiments, an applied potential of −100 mV vs. Ag/AgCl is chosen since high currents can be achieved, here, at relatively low overpotentials. Additionally, this potential enables good comparability to previous studies. 37,58 3.4 Verification of the dyes in the photocurrent measurement Photo-action spectroscopy has been executed next, to prove that the presence of the fluorophores leads to a better performance of the constructs in the green gap. Therefore, the photobiocathodes have been illuminated with light of different wavelengths. The results of these experiments are compiled in Fig. 4 and S5. † For the PSI-ATTO590 construct, FRET has been reported before, 56 but here it can be demonstrated that this can be exploited beneficially for the current generation in photobiocathodes. Interestingly, also for the PSI-ATTO532 variant, an additional photocurrent compared to electrodes with native PSI alone can be obtained at around 540 nm. Fig. 4 Photo-action spectra of the photobiocathodes based on the different constructs. (A) MET with cyt c and the constructs on 3D ITO in 5 mM PPB pH 7; (B) DET without a mediator in 100 mM MES pH 6 and 400 mM KCl. When both dyes are coupled to PSI (PSI-ATTO-mix), it can generate photocurrents in the wavelength range of both fluorophores. Such photocurrent can be seen for electrodes with cyt c (MET) as well as electrodes based on DET. In conclusion, one can state that the presence of the fluorophores can be verified on a functional level. It results in an improved photocurrent generation in the green gap compared to unmodified PSI. This finding corresponds well to concepts in the literature. 17,36,39,57 3.5 Photocurrent response As stated previously, the main goal of the application of the fluorophore-modified constructs instead of native PSI is the improvement of the performance of the photobiocathodes. Hence, after the proof of principle that the dyes can be seen in the green gap during photo-action spectroscopy (Section 3.5), photocurrents have been measured. Like before, 3D ITO electrodes of about 20 μm thickness and a white light source at 100 mW cm −2 have been used for all experiments. A constant potential of −100 mV vs. Ag/AgCl is applied as discussed in Section 3.3. 3.5.1 Photobiocathodes based on cyt c as the mediator The photocurrent densities for PSI and the three constructs are depicted in Fig. 5A . For unmodified PSI, photocurrents of −32.5 ± 3.6 μA cm −2 have been obtained for electrodes based on MET. These numbers correspond well to the literature for such a system. 37 Fig. 5 Performance of the biohybrid setups prepared by the different constructs. Pure: PSI-pure; ATTO590: PSI-ATTO590; ATTO532: PSI-ATTO532; ATTO-mix: PSI-ATTO-mix. (A) Photocurrent of the photobiocathodes relying on MET and DET. Pure: MET ( n = 4) and DET ( n = 7); ATTO590: MET ( n = 4) and DET ( n = 4); ATTO532: MET ( n = 4) and DET ( n = 11); ATTO-mix: MET ( n = 4) and DET ( n = 3); (B and C) representative photocurrent measurements for (B) DET and (C) MET respectively. Photocurrent measurements conducted in buffer F for DET and buffer E for MET at −0.1 V vs. Ag/AgCl and 100 mW cm −2 illumination. The performance of all constructs differs significantly from that of the non-modified reference. The PSI-ATTO590 construct works drastically better than the PSI alone (−55.3 ± 7.0 μA cm −2 ). The PSI-ATTO532 variant performs slightly better but is still within the error bars of both values (−60.9 ± 9.5 μA cm −2 ). The PSI-ATTO-mix construct also shows a significant photocurrent increase (−50.1 ± 6.4 μA cm −2 ) compared to electrodes with native PSI, but no further enhancement is achieved compared to the constructs with only one fluorophore. These findings show a comparatively large enhancement when the absorption spectra of PSI and the constructs are considered only, but they match well with the activities of natural and modified PSI (measured by oxygen consumption in solution – see Section 3.1), which has been found enhanced for all three constructs by a factor of about 2. This means that the enhanced activity found in solution (which includes not only the light interaction but also the cyt c reaction) is well reflected in the electrode performance of the constructs with respect to the non-modified PSI. 3.5.2 Photobiocathodes based on DET from the ITO electrode The evaluation of DET-based electrodes and, thus, systems avoiding an additional component exhibits more remarkable differences in the performance caused by the various constructs. Photobiocathodes based on native PSI achieve photocurrents of −8.5 ± 0.8 μA cm −2 , which matches well with the literature. 58 Interestingly, electrodes with the PSI-ATTO590 construct do not provide a higher photocurrent compared to this unmodified reference (−7.7 ± 1.0 μA cm −2 ). Furthermore, their current response kinetics is much slower than for PSI and the other variants, as illustrated in Fig. 5B . In contrast, the PSI-ATTO532 variant shows some extraordinary effects within the 3D ITO electrode structure: the photocurrents are almost 6-fold higher for this construct (−47.0 ± 7.4 μA cm −2 ) compared to unmodified PSI. This increase surpasses the increment observed in the UV/vis study as well as in the activity measurements (Section 3.1) and, thus, cannot be explained by the additional absorption of light in the green gap alone. An important hint can be obtained when the non-normalized photo-action spectra are compared (Fig. S6 † ). Here, the photocurrent enhancement occurs in the whole wavelength range studied. This means that even for wavelengths, which are not absorbed by the fluorophore, the photocurrent is significantly higher than for the other electrodes. Photobiocathodes using PSI-ATTO-mix exhibit an intermittent behavior compared to PSI-ATTO590 and PSI-ATTO532 electrodes (−30.4 ± 3.0 μA cm −2 ). Previously, it was already verified that without PSI no significant photocurrents can be obtained on bare nanoparticular 3D ITO electrodes. 58 To rule out that the dyes interacting with the artificial surface define the photocurrent output, control measurements with the dyes only have been conducted. The obtained current responses are depicted in Fig. S7. † No clear photocurrent signals can be determined. This demonstrates that PSI is the photoactive component responsible for photocurrent generation, which is influenced by the coupled dyes as illustrated in Fig. 2 . 3.5.3 Comparison of MET and DET modes While the photocurrent density is in general significantly higher for MET, also other differences appear between the two electron transfer mechanisms. As just discussed, the kinetics for the DET to PSI-ATTO590 is poor, whereas the current response is very fast for the other systems. Here, when no mediator is present, the maximal photocurrent is reached within the first second after switching-on the light. In contrast, for MET the kinetics is slower but similar for all variants. It takes about 5 s to reach the maximum current density as depicted in Fig. 5C . As shown, the covalent binding of fluorophores to PSI changes its optical properties but has also other effects that are essential when applying such constructs in photobioelectrodes. Two main reasons can be seen for the different photocurrent output of electrodes in both MET and DET modes: the coupling of dyes to the PSI surface can impact the number of immobilized protein complexes and their orientation to the ITO surface. While the first aspect directly influences MET- and DET-based electrodes, the latter is of higher relevance for electrodes in DET mode. Thus, the number of immobilized biomolecules has been studied and will be discussed in the next section with respect to the photocurrent behavior illustrated above. 3.6 Protein coverage on the photobioelectrodes The coverage with biomolecules has been determined to trace possible causes for the performance differences of the constructs. For this purpose, chlorophyll has been extracted from the electrodes after preparation like described in Section 2.5. As can be seen in Fig. 6A , significant differences appear. It must be noted that the electrode preparation has not been identical for photobiocathodes operating in MET and DET modes (see Section 2.3). Fig. 6 Comparison of the biocomponents in the photobiocathodes. (A) PSI and cyt c coverage of the structures; for electrodes in DET mode the only present biomolecule is PSI (black dots), and in the case of MET-based electrodes, two numbers – for PSI (red squares) and cyt c (red crosses) – are given; n = 4 for PSI (DET) ATTO590 and ATTO-mix, and n = 3 for the remaining; (B) turnover frequencies of the diverse electrodes calculated by using the values of Fig. 5A and 6A For MET, after the incubation with PSI, cyt c is applied. This additional incubation in a highly concentrated cyt c solution can disrupt the binding of PSI to the ITO surface. Here, the unmodified PSI shows the highest adsorption to the ITO surface, whereas the PSI constructs result in somewhat smaller surface concentrations. When the cyt c coverage is analyzed, similar electro-active concentrations have been found, with a small peak for electrodes with the PSI-ATTO532 variant. Since the number of bound PSI constructs is diminished for electrodes operating in MET mode (compared to the native protein complex), but the photocurrents are found to be larger, a significantly higher turnover number can be elucidated for the electrodes with the fluorophore-coupled PSI. The highest values are obtained here for the PSI-ATTO532 and the PSI-ATTO-mix construct (105 e − /PSI/s). However, in the latter case the amount of immobilized photoactive protein is the smallest; consequently, the overall photocurrent enhancement is the lowest among the constructs (although significant). Evaluating the electrodes prepared in DET mode, again the highest surface concentrations have been found for the native protein complex (18.4 ± 1.0 pmol cm −2 ). Slightly smaller values are obtained for PSI-ATTO532 and PSI-ATTO-mix, 15.8 ± 1.3 pmol cm −2 and 14.7 ± 0.8 pmol cm −2 , respectively. The PSI-ATTO590 loading falls out of line. Here, just 7.8 ± 0.4 pmol cm −2 is bound to the ITO electrode material which is a factor of 2 lower than that for the other constructs. This low coverage can explain the poor performance of the PSI-ATTO590 construct on the 3D ITO in DET mode. When the turnover frequencies (TOFs) are compared ( Fig. 6B ), it stands out that this variant performs about 2 times better than the reference specimen, 10 vs. 5 e − /PSI/s. This is in line with the oxygen consumption activity (Section 3.1), where the same difference in the turnover rate appears. The highest TOF is obtained for the PSI-ATTO532 modification with 31 e − /PSI/s, which is a factor of 6.5 better than that of the natural PSI. The value for PSI-ATTO-mix lies between the others at 21 e − /PSI/s. The disparity between the constructs might be explained by the chemical structure of the dyes. Although they are rather similar, there are some differences that are necessary to obtain different optical properties. ATTO 590 consists of 5 condensed aromatic rings; thus, it is hydrophobic. In ATTO 532, two sulfo groups provide a higher hydrophilicity and a negative net charge. These differences could lead to some consequences: both dyes bind in different areas of the PSI molecules and/or they change the binding behavior of the modified PSI to the ITO structure, i.e. , the number and orientation, of the adsorbed constructs. The former is a probable explanation for the PSI-ATTO590 electrode behavior since here less construct binds to the ITO structure. In contrast, PSI-ATTO532 performs better than envisioned although the PSI loading is not enhanced compared to the unmodified protein complex. Hence, the most likely reason for the better output is that the ATTO 532 dyes aid in an accurate orientation of PSI towards the ITO nanoparticular surface. This conclusion originates from the fact that a rather similar amount of the photo-active protein is immobilized in the 3D ITO for the native PSI and the PSI-ATTO532 construct, but a significantly higher photocurrent is observed for the modified protein. This means that a larger fraction of the photoactive complexes is in a productive orientation with respect to the ITO surface for the construct-based electrode. This explanation is strongly supported by the finding that the photocurrent with the ATTO532 construct is enhanced in the whole wavelength range used in photo-action spectroscopy, i.e. , even at wavelengths where this dye does not absorb light (Fig. S6 † ). In summary, one can state that the fluorophore modification helps not only in photon collection, but also in optimizing the interaction of the photoactive protein complex with the electrode surface. The intermittent behavior of PSI-ATTO-mix seems to be consistent with the discussed effects for attached ATTO 532 and ATTO 590 for the interaction with the ITO structure. Further studies will, however, be necessary to elucidate the details of the fluorophore coupling to PSI and the interaction of the resulting constructs with the ITO surface. 3.7 Evaluation of the PSI-ATTO532 photobiocathode Since the PSI-ATTO532 construct outperforms the other variations in DET mode on 3D ITO and the expectations from the additional light interaction in the green gap, further measurements have been conducted to accumulate more information about this photobiocathode. First, the photocurrent response at different illumination intensities has been evaluated. So far, all experiments have been executed at 100 mW cm −2 , which roughly corresponds to the power of solar radiation. Now, the illumination intensity has been reduced stepwise by 3 orders of magnitude down to 0.1 mW cm −2 . The results are depicted in Fig. 7A . An almost linear increase of the current response with the light intensity is obtained even for high intensities, which is remarkable. Fig. 7 Photocurrent measurements of photobiocathodes without a mediator in 100 mM MES pH 6 and 400 mM KCl at an applied potential of −100 mV vs. Ag/AgCl. (A) Comparison of the performance of PSI-ATTO532 ( n = 4) and native PSI ( n = 3) at different light intensities; (B) behavior over two hours of 3 electrodes (mean value) prepared with PSI-ATTO532. Another critical aspect of photobioelectrodes is their stability under repeated illumination. Thus, 30 light pulses with a duration of 2 min each have been applied onto PSI-ATTO532 photobioelectrodes. The photocurrent response is relatively stable, but tends to decrease over time, as illustrated in Fig. 7B and S8. † After two hours of light treatment, a total of 60 min illumination, the initial performance is halved. This is significantly better than findings in many previous studies when usually 50% of the starting value is reached after 1/4 th of this time period. 38,41,63 However it has to be noted that the reported photobioelectrodes have been relying on MET. Better stability has been found for DET-based electrodes with unmodified PSI. 58 However, in the present study, much higher photocurrents have been obtained with the fluorophore-modified PSI so that even after more prolonged operation, much higher photocurrent values can be retained compared to the unmodified PSI. 3.8 Classification of the findings As stated in the Introduction, only a few studies have been undertaken with modified biomolecules to close the green gap in photobioelectrodes. Light-harvesting bacterial reaction center core complexes were modified with four different dyes (Alexa 647, Alexa 680, Alexa 750, and ATTO 647N) and coupled to ITO electrodes. By that, 1.6 and 1.8 μA cm −2 were achieved, 36,57 which is 35 times less than that obtained in this study. Takekuma et al. combined TiO 2 , PSI, and a perylene di-imide derivative to measure photocurrents up to 430 μA cm −2 . 39 Yet, it must be noted that most photocurrent is already available without the photosystem. Since the electrolyte contained guanidine thiocyanate, it is also questionable whether the proteins were still intact during measurement. Additionally, no photo-action spectra or similar experiments are shown to verify PSI as the current source. Some photobioelectrodes applying non-modified PSI have been reported in the literature and can be used for comparison with these novel photobiocathodes to some extent. However, most of the studies have been using a mediator to improve the performance. As stated in Section 3.5, the values obtained for the reference specimen (native PSI) compare well to earlier results with the same electrodes. 37 Hence, the photocurrent output, up to −60.9 ± 9.5 μA cm −2 , of the fluorophore-modified electrodes is 2-times higher than that for natural PSI-based ones under similar conditions (here, MET mode with cyt c as the immobilized mediator). This clearly demonstrates the potential of improving the spectral properties of PSI for enhanced light-to-current conversions. The measurements demonstrate that besides the optical properties, the binding to the surface can be impacted by the fluorophore coupling. This can also compensate for improved light interactions, as shown for electrodes with PSI-ATTO590 and cyt c as mediator. There is also a significant development in new electrode materials. For example, a more transparent ITO electrode can be prepared when starting from solutions of precursor compounds instead of nanoparticles as done in this study. Such electrodes have already resulted in much higher photocurrents. 38 Thus, this will open more opportunities for using fluorophore-coupled PSI to increase photocurrent output further. In the literature, not many values obtained by DET are available for comparison. However, in a previous study, 10.1 μA cm −2 was achieved for a similar electrode after an intensive evaluation of the measurement conditions. 58 This benchmark is outperformed by a factor of 5 by PSI-ATTO532 with even thinner ITO electrode structures. Here it can be stated that besides the filling of the green gap of PSI, mainly a better interaction of the luminal side with the ITO surface contributes to the substantial performance increase while retaining the adsorption capability of the PSI construct to the 3D ITO material. To the best of our knowledge, 47.0 μA cm −2 is the record value for DET of any photoactive biomolecule on a 3D material. The previous record – 33 μA cm −2 set for PSII on 3D ITO is surpassed by a factor of ca. 1.5. 64 An interesting aspect is the comparison of the performances of electrodes based on the two evaluated electron transfer mechanisms. When similar photobioelectrodes have been tested before, DET has often been found to be far more than one order of magnitude less effective than MET. 9,21,37,38,41 Although the direct interaction between ITO and PSI has been thoroughly studied recently and parameters have been optimized, 58 this pathway is still about a factor of 4 less effective for natural PSI as shown in the present study (−8.5 compared to −32.5 μA cm −2 in MET). However, owing to coupled ATTO 532, this gap narrows drastically so that the mediator-less system achieves more than 3/4 of photocurrent density of the setup with incorporated cyt c (−47.0 vs. −60.9 μA cm −2 ). Furthermore, these new photobiocathodes based on DET and PSI-ATTO532 outperform the established mediator-based electrodes with natural PSI and cyt c by about 50% (−47.0 vs. −32.5 μA cm −2 )."
} | 10,632 |
26629901 | PMC4668087 | pmc | 6,223 | {
"abstract": "Genome-scale metabolic models usually contain inconsistencies that manifest as blocked reactions and gap metabolites. With the purpose to detect recurrent inconsistencies in metabolic models, a large-scale analysis was performed using a previously published dataset of 130 genome-scale models. The results showed that a large number of reactions (~22%) are blocked in all the models where they are present. To unravel the nature of such inconsistencies a metamodel was construed by joining the 130 models in a single network. This metamodel was manually curated using the unconnected modules approach, and then, it was used as a reference network to perform a gap-filling on each individual genome-scale model. Finally, a set of 36 models that had not been considered during the construction of the metamodel was used, as a proof of concept, to extend the metamodel with new biochemical information, and to assess its impact on gap-filling results. The analysis performed on the metamodel allowed to conclude: 1) the recurrent inconsistencies found in the models were already present in the metabolic database used during the reconstructions process; 2) the presence of inconsistencies in a metabolic database can be propagated to the reconstructed models; 3) there are reactions not manifested as blocked which are active as a consequence of some classes of artifacts, and; 4) the results of an automatic gap-filling are highly dependent on the consistency and completeness of the metamodel or metabolic database used as the reference network. In conclusion the consistency analysis should be applied to metabolic databases in order to detect and fill gaps as well as to detect and remove artifacts and redundant information.",
"introduction": "Introduction Metabolic reconstruction is the computational process that aims to elucidate the biochemical network of reactions and metabolites which defines the cell metabolism of a certain organism [ 1 , 2 ]. Since metabolic reconstruction is tightly integrated with genomic information, it can be viewed as a detailed functional annotation of the genome [ 3 , 4 ]. In the first stages of a reconstruction, the genome sequence and its annotation are the main source of information used to infer the biochemical pathways of an organism [ 5 ]. Furthermore, each entry annotated as an enzyme coding gene usually contains some identifiers, such as Gene Ontology (GO) terms or Enzyme Commission (EC) numbers, which allow the construction of the gene-protein-reaction rules [ 6 ], by mapping one or more coding sequences to one or more reactions, through a protein or protein complex. After this, a metabolic database is used to map the enzymatic activities to instances of biochemical reactions, through their EC numbers [ 7 ]. A metabolic database typically describes collections of enzymes, reactions and biochemical pathways, which cover most of the known biochemistry [ 8 , 9 ]. Databases commonly used in metabolic reconstruction include the SEED [ 10 ], BiGG [ 11 ], KEGG [ 9 ] or Metacyc [ 12 ], among others. Although the objective of a metabolic reconstruction may be to create an organism's specific metabolic database, in many cases the final goal is to develop a genome-scale metabolic model (GSM), that is to say, an in-silico representation of a metabolic network [ 13 ]. A GSM can be used to generate hypotheses about the metabolic capabilities of the network through the computational framework known as constraint-based modeling (CBM), which eventually may be experimentally tested [ 14 , 15 ]. Genome-scale reconstruction has rapidly grown in recent years, as has its range of applications [ 16 , 17 ]. Moreover, CBM can be used to improve model formulation by the detection and resolution of inconsistencies. In this sense the analysis of a GSM can be used to refine the annotation of the genome [ 18 , 19 ] and thus to improve model formulation. In general, inconsistencies will appear as holes in the structure of the network. These holes might indicate either global or organism specific gaps in biological knowledge. Global gaps are reflected in the existence of metabolites with an unknown biochemical fate [ 20 ], as well as the large number of orphan enzymatic activities [ 21 , 22 ]. On the other hand, organism specific gaps are commonly associated with genome annotation errors reflected in the absence of enzymatic activities coded in the genome, or in the inclusion of activities that are not present in the considered metabolism [ 23 ]. The resolution of inconsistencies in GSMs is known as network curation. This is a decision- making process where wrongly annotated reactions are removed and candidate reactions are included for the purpose of solving model gaps. The prediction of candidate reactions for filling gaps is referred to as the gap-filling problem [ 23 , 24 ]. Several methods have been proposed for identifying and solving inconsistencies in GSMs in an automatic fashion. Some of these methods rely on the application of optimization techniques [ 18 , 24 – 29 ], while others focus on a genomic approach to find missing genes [ 30 , 31 ]. In general, these methods require a metabolic database to search for candidate reactions. However, in optimization-based methods the database itself is treated as a large metabolic model where the GSM to be gap-filled is embedded [ 18 , 32 ]. Since metabolic databases include, in general, reactions that span across the tree of life, they can be considered as global networks or metamodels [ 33 ]. The analysis of a metamodel through the CBM approach can lead to the detection of structural inconsistencies, as well as coupling relations [ 34 ], such as enzyme subsets [ 35 ]. Remarkably, the presence of inconsistencies in a metamodel will be propagated to any particular GSM derived from it. As a consequence, the consistency of the metamodel is of critical importance in order to reconstruct a consistent GSM. On the other hand, the information derived from the coupling relations present in a metamodel can facilitate the reconstruction and curation of GSMs. Previous studies have focused on the use of a global network to improve the reconstruction of single organisms [ 33 ], although a graph-based approach was there adopted rather than the constraint-based modeling [ 36 ]. The aim of the large-scale analysis proposed in this paper is to detect both the recurrent errors in the automatic reconstruction process as well the sources of these inconsistencies, and to assess the impact of the metamodel completeness and consistency in the gap-filling process. To this end, a dataset of 130 GSMs of bacteria reconstructed using the Model SEED pipeline [ 37 ] was selected for this study, from which a metamodel was formulated. The main reason for choosing the mentioned dataset relies on the fact that the SEED is a wide extended pipeline for metabolic reconstruction. Additionally, it has been used in several works to perform large scale studies of bacterial metabolism, and to draw important biological conclusions [ 38 – 42 ]. A consistency analysis was performed on the metamodel using the unconnected modules approach [ 43 ]. The detected inconsistencies were manually resolved using information from the following metabolic databases: SEED [ 10 ], KEGG [ 9 ] and Metacyc [ 12 ]. The resulting consistent metamodel was used as reference to solve the gap metabolites and blocked reactions in each individual GSM, using a modified version of the fastcore algorithm [ 26 ]. Moreover, each GSM was also gap-filled using the initial metamodel ( i . e . without manual curation). A comparative analysis was performed between both versions of each GSM to investigate the impact of perform gap-filling using an inconsistent reference network. Finally, a set of models of certain organisms not considered during the construction of the metamodel was used, as a proof of concept, to extend the metamodel with new biochemical information, and asses its impact on the gap-filling results. Background Any biochemical network such as cellular metabolism can be represented by its corresponding stoichiometric matrix N \n mxn . This matrix, where rows and columns correspond to metabolites and reactions respectively, represents the structure of the network [ 44 ]. Moreover, the set of reactions J indexes can be partitioned into two disjoint subsets: 1) the set J \n INT of biochemical reactions that take place inside the cell, as well the transport reactions that operate between the cell and the surrounding medium; 2) the set of exchange fluxes J \n EX , which are auxiliary variables used to represent the rate at which the metabolites that are allowed to cross the system boundary are consumed/produced [ 45 ]. The structural properties of such networks may be assessed through the CBM approach. CBM relies on the use of constraints with the aim of reducing the system's functional states, to those that are physiologically more relevant [ 14 , 46 ]. Two fundamental constraints, and a set of reaction bounds, are used to define the so-called flux space. First, the steady state assumption, imposed over the mass balance equation of each metabolite of the network:\n N ⋅ v = 0 (1) \nwhere v is the vector of reaction fluxes, or flux distribution. Second, the thermodynamic constraints, which ensure that the irreversible reactions take non-negative flux values.\n 0 ≤ v j ∀ j ∈ I r r (2) \nwhere Irr is the set of the irreversible reaction indexes. Besides, lower and upper bounds are imposed over each reaction to represent additional constraints, such as the maximum capacity of an enzyme and also to model the surrounding environment of a metabolic system, i . e . by constraining the exchange fluxes J \n EX :\n β i ≤ v j ≤ α i ∀ j ∈ J (3) \nwhere β \n i and α \n i correspond to the lower and upper bound of reaction i , respectively. \n Definition: Any non-trivial vector v feasible with respect to Eqs ( 1 ) and ( 2 ) is called a flux distribution. The set of all possible flux distribution spans the so-called flux space:\n F = { v → ∈ R n : N ⋅ v → = 0 , β i ≤ v j ≤ α i ∀ j ∈ J , β i = 0 ∀ i ∈ I r r } (4) \n An important step in the reconstruction of a metabolic model is to check the consistency of the network definition. In general terms, the structural inconsistencies of a metabolic model may manifest as blocked reactions, i . e . reactions which cannot display a steady-state flux other than zero, and gap metabolites, i . e . nodes in the network through which there can be no steady state flow [ 24 ]. Formally speaking, given a metabolic model with stoichiometric matrix N and a set of irreversible reactions Irr , structural inconsistencies are defined as follows: \n Definition: a reaction j is defined as blocked if for every possible flux distribution v the corresponding flux value v \n j = 0 . \n Definition: a metabolite is defined as a gap if all the reactions in which it participates are blocked. Moreover, the relation between blocked reactions and gap metabolites can be unambiguously established by means of the unconnected modules [ 43 ], as following defined: \n Definition: an unconnected module (UM) is defined as a connected bipartite sub-graph of blocked reactions connected through gap metabolites. \n Definition: a metabolic model with a stoichiometric matrix N and a set of irreversible reactions Irr is said to be flux consistent if it does not contain blocked reactions nor gap metabolites.",
"discussion": "Discussion Finding structural inconsistencies in GSMs The consistency analysis performed on a dataset of 130 GSMs of different bacteria showed that most of the models have at least 25% of their reactions blocked. The results also showed that the reconstruction of a non-model organism tends to become easier when the GSM of a phylogenetically closer organism exists, as previously discussed [ 16 ]. In the analyzed dataset, this is reflected by the fact that the models with less than 25% of their reactions blocked belong to the class of gammaproteobacteria , which includes E . coli . As expected, the model with the lowest percentage of blocked reactions was the one corresponding to E . coli K12 (Opt83333.1) with a value of ~19%. The same measure was also evaluated for the last version of the manually reconstructed GSM of E . coli K12, named iJO1366 [ 57 ] , and the result showed that 10% of its reaction were blocked. The difference found was expected, since iJO1366 has been extensively manually curated, whereas Opt83333.1 was automatically reconstructed. On the other hand, T . thermophilus (Seed300852.3) was shown to be the model with the highest percentage of blocked reactions (~47%). As in the case of E . coli , this measure was compared against the value obtained for a recently published GSM of T . thermophilus , named iTT548 [ 62 ], where ~27% of its reactions were found blocked. This value, although high, shows that manual curation dramatically improves the consistency of a GSM. Furthermore, the use of additional experimental data will also help to improve the consistency and completeness of a GSM. Gap-filling through metamodel analysis A comparative analysis surprisingly showed that 480 reactions (~22%) are blocked in each GSM where they are present ( i . e . “always blocked”). This showed a remarkable number of network inconsistencies at the level of the metabolic database used as a reference during the reconstruction process. When these reactions were grouped by subsystem, 25% were found to be involved in the biosynthesis of cell wall components as well as membrane lipids (see S2D Table ). This showed that the automatic inferences of such metabolic pathways is particularly hard because of its iterative nature, along with the diversity of cell envelope components, and the absence of experimental information about cell membranes for most of the organisms analyzed. Furthermore, pathways for the biosynthesis of important cofactors, such as siroheme, biotin and vitamin B12, were also found to be always blocked. In some cases the pathway was incomplete whereas in other cases it was complete but blocked. For example, the curation of the biotin biosynthesis is an illustrative example of how models must evolve in parallel to biochemical knowledge (see Construction and curation of the metamodel in result section). The detection of recurrent inconsistencies motivated a global consistency analysis, i . e . the analysis of the global metabolic network. In the present paper a metamodel, named MM130.0, including 130 GSMs was reconstructed using the SEED pipeline. The consistency analysis and the visual inspection of the set of UMs founded in MM130.0 allowed: first, the detection of reactions wrongly included during the reconstruction of the individual GSMs ( e . g . some activities belonging to methanogenesis); second, the detection and resolution of incomplete pathways by the addition of the corresponding missing reactions, e . g . the pimelic acid biosynthesis ( i . e . the first stage of biotin biosynthesis); third, the detection and resolution of global deadend metabolites such as the cases of S-adenosyl-4-methylthio-2-oxobutanoate and of some biomass components not previously included in the biomass reaction of any GSM. The curated metamodel MM130.1 can be used as a reference network for the curation of any model generated using the SEED. In this way, different algorithms and weights can be tested for the curation of a particular model. Additionally, the metamodel presented here can be modified or extended to adjust to particular situation such as the case of organisms with highly reduced genomes. Impact of metamodel consistency on automatic gap-filling Many gap-filling algorithms have been developed by focusing on the structure of the objective function. However, the possible impact of the reference network consistency, which affects the gap-filling process, has received little or no attention. Therefore, a gap-filling was performed on the whole dataset to evaluate the impact of the metamodel completeness and consistency, using alternatively MM130.0 and its curated version MM130.1. When the reaction content was compared within each pair of curated GSMs, in 125 of the cases the version gap-filled with MM130.1 contained 85% or more of the reactions present in the version gap-filled with MM130.0. Strikingly, in five models the percentage of inclusion varies between 65–80%, which indicates a remarkable difference between the two versions. Thus, it can be concluded that in some cases the result of an automatic gap-filling can be highly dependent on the metamodel used as the reference. In particular, the divergence was found to be greater for the smaller models. Since, these organisms correspond to obligate parasite and symbionts, their metabolisms tend to lack many biosynthetic capabilities and thus, they should import a great number of metabolites from the surrounding environment. Furthermore, the possibility of a metabolic complementation between parasites or symbionts and their hosts may result in the exchange of metabolites not commonly included in growth media. Thus solving such situation requires a manual inspection of individual UMs to evaluate the possibility of adding unknown transporters and the corresponding exchanges fluxes [ 43 ]. As a consequence, the metabolic reconstruction of such organisms is more susceptible of gap-filling artifacts Finally, the gap-filling results showed that in many cases the number of orphan gap-filling reactions included to solve gaps significantly exceeds the number of gaps. This result is independent of the metamodel version used, and suggests that in many situations blocked reactions can be artifacts and should be removed from the model instead of adding new reactions to solve what at first looked like a gap. For example, the initial GSM of M . genitalium G-37 (Opt243273.1) contained 292 reactions, of which 118 were blocked, whereas both gap-filled versions included more than 500 reactions. The manually reconstructed model of M . genitalium G-37 iPS189 [ 28 ] contains 265 reactions, of which 90 are orphans, which indicates that gap-filled version of Opt243273.1 significantly overestimates the number of reactions. Such kinds of discrepancies clearly indicate that, in this case, most of the blocked reactions should be pruned rather than gap-filled. This result showed that the curation of a metamodel is necessary, but not sufficient condition, in order to perform automatic model curation. The extension of MM130.1 to MM166.1 by the addition of 36 GSMs allowed us to show how a metamodel can be updated to include new biochemical information. In this particular case the inclusion of 36 additional models provided only 141 reactions and 55 and transporters, and most of these reactions belong to a single organism ( P . difficile ). Nevertheless, this new information was enough to affect the outcome of the gap-filling process, as has been shown (see section Extending a metamodel with new biochemical information ). Moreover, the increase in fastcore' s efficacy when the expanded metamodel was used was expected, taking into account the fact that fastcore finds a minimal consistent model by minimizing the number of reactions added to solve inconsistent reactions. Consequently, if new pathway branches are available due to the utilization of a bigger metamodel, an improvement in the achieved minimum is possible. However, this minimization criteria may lead to artifacts such as chimerical pathways composed of reactions of organisms not related to the one being analyzed. Taking everything into account, it is worth to note that the automatic reconstruction and curation of metabolic models should consider the taxonomic distribution of the reactions, so that the weights assigned to them can be properly adjusted when formulating the gap-filling problem, as has been discussed by other authors [ 29 , 63 ]. The reconstruction of a GSM depends, to a great extent, on the quality of the genome annotation. Thus, errors in the annotation will lead to the presence of inconsistencies in the model formulation. In this context, in principle there are two different types of annotation error: 1) the absence of certain functional annotations, e . g . an enzyme coding gene not annotated as such; 2) the incorrect assignment of an enzymatic activity to certain coding sequences. The presence of type 1 and 2 errors will produce the absence, or the wrong inclusion, of some reactions in the GSM, respectively. Thus, gaps in a GSM caused by distinct types of errors will require a differential treatment. In the case of type 1, missing reactions should be gap-filled. However, in the case of type 2 errors, the wrong added reactions should be removed. Since both types of errors will manifest as blocked reactions and gap metabolites, we argue that the problem resides in how to differentiate, in an automatic fashion, between the blocked reactions that have to be gap-filled (in order to restore their connectivity) and the erroneous reactions that should be removed. As shown in this paper the distinction between these types of errors is far from trivial, and not considering them will result in the overestimation of the reaction content of an organism. Although there have been considerable advances in the automatic reconstruction of GSMs, the outputted models still contain many structural inconsistencies, as the results presented have shown. Furthermore, the detection of recurrent inconsistencies manifested as metablocked reactions indicates that gaps may already be present in the metamodel or the metabolic database used to perform the reconstructions. As a consequence, we argue that consistency analysis, such as the one presented in this paper, should be applied to metabolic databases in order to detect and fill gaps as well as to remove artifacts and redundant information."
} | 5,492 |
32526923 | PMC7356029 | pmc | 6,224 | {
"abstract": "Arbuscular mycorrhizal fungi (AMF) have been shown to play an important role in increasing plant fitness in harsh conditions. Therefore, AMF are currently considered to be effective partners in phytoremediation. However, AMF communities in high levels of petroleum pollution are still poorly studied. We investigated the community structures of AMF in roots and rhizospheric soils of two plant species, Eleocharis elliptica and Populus tremuloides , growing spontaneously in high petroleum-contaminated sedimentation basins of a former petrochemical plant (91,000 μg/Kg of C10–C50 was recorded in a basin which is 26-fold higher than the threshold of polluted soil in Quebec, Canada). We used a PCR cloning, and sequencing approach, targeting the 18S rRNA gene to identify AMF taxa. The high concentration of petroleum-contamination largely influenced the AMF diversity, which resulted in less than five AMF operational taxonomical units (OTUs) per individual plant at all sites. The OTUs detected belong mainly to the Glomerales, with some from the Diversisporales and Paraglomerales, which were previously reported in high concentrations of metal contamination. Interestingly, we found a strong phylogenetic signal in OTU associations with host plant species identity, biotopes (roots or soils), and contamination concentrations (lowest, intermediate and highest). The genus Rhizophagus was the most dominant taxon representing 74.4% of all sequences analyzed in this study and showed clear association with the highest contamination level. The clear association of Rhizophagus with high contamination levels suggests the importance of the genus for the use of AMF in bioremediation, as well as for the survey of key AMF genes related to petroleum hydrocarbon resistance. By favoring plant fitness and mediating its soil microbial interactions, Rhizophagus spp. could enhance petroleum hydrocarbon pollutant degradation by both plants and their microbiota in contaminated sites.",
"introduction": "1. Introduction Arbuscular mycorrhizal fungi (AMF) are obligatory fungal symbionts forming a symbiosis with up to 80% of land plant species on earth [ 1 , 2 ]. As mutualists, AMF generally improve plant growth by increasing their uptake of mineral nutrients, in particular, phosphorus [ 3 , 4 , 5 ]. As a trade-off, AMF receive carbon from the plant [ 6 ]. In addition to their role in plant nutrition, multiple studies have shown that AMF enhance plant survival from biotic and abiotic stress such as nutrient limitations, fungal and bacterial plant pathogens, plant-parasitic nematodes, salinity, drought, trace elements and petroleum hydrocarbon pollutants [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. The crucial roles of AMF in the survival of host plants in harsh environments have led researchers to consider them as useful partners in phytoremediation. To date, a number of studies have shown that AMF could improve phytoremediation processes to clean up soil polluted with trace metals [ 8 , 15 , 16 , 17 ]. Many studies have also indicated that the application of AMF can impact the community structure of soil microbes that enhance degradation, sequestration or stabilization of pollutants [ 18 , 19 ]. However, the mechanisms through which AMF can favor phytoremediation are yet to be understood. Moreover, multiple variables can affect the AMF community structure, especially in field conditions. AMF were shown to exhibit host-plant dependency and a community structure that varied in different environmental conditions [ 20 , 21 , 22 , 23 ]. Previous studies also indicated that the diversity of AMF could be modified by contaminants such as trace elements and petroleum hydrocarbons [ 19 , 24 , 25 , 26 ]. These various factors affect AMF community structure and make the application of AMF in the field challenging and unpredictable. In addition, studies on remediation of petroleum hydrocarbons by the use of AMF in large-scale trials remain to be conducted. By better understanding the dominant AMF taxa in petroleum hydrocarbon contaminated sites, we could harness these taxa, which potentially exhibit high tolerance to contamination stress, and use them as bioinoculants in phytomanagement. Therefore, it is essential to increase our knowledge of the effect of biotic and abiotic environmental factors on AMF communities in highly petroleum hydrocarbon contaminated sites to design successful phytoremediation strategies. At the same time, multiple genome sequencing projects on AMF suggest different phylogenetic clades of AMF can have dramatically different gene repertoires for their adaptation and functioning in ecosystems [ 27 , 28 ]. Thus, if there is strong phylogenetic signal observed among AMF under high contamination, it is likely that the conserved genes of certain clades are related to the distribution. To date, it has not been clearly understood whether certain AMF clades exhibit high tolerance against high petroleum hydrocarbon contamination. Many papers on AMF have reported the occurrence of members belonging to the genus Rhizophagus [ 24 , 26 ], which was considered to be an important genus because of its dominance in both inorganic and organic polluted soils. The objectives of this study were (1) to investigate the diversity and community structures of AMF associated with two native plant species growing spontaneously in high petroleum hydrocarbon polluted environments; and (2) to evaluate the effect of contaminant concentrations on AMF community structure. Specifically, we hypothesized that (1) plants recruit different AMF communities under different concentrations of petroleum hydrocarbon, and those communities exhibit some dominant taxa, particularly in highly contaminated sites; (2) contamination concentration shapes AMF community structure much more strongly than host identity and biotopes. To address these objectives and test the hypotheses, we used PCR, cloning and Sanger sequencing based on the 18S rRNA gene to amplify an approximately 750 bp fragment from AMF. We report the correlations between the AMF taxa and three distinct environmental factors: host plant species identity, biotopes (roots or rhizospheric soils), and contaminant concentrations (lowest, intermediate and highest).",
"discussion": "4. Discussion The diversity of plant species found at a site can be strongly influenced by concentrations of inorganic and organic contaminants [ 41 , 42 , 43 ]. Due to the high concentrations of petroleum hydrocarbon contamination ( Supplementary Table S1 ) that greatly affected the diversity and distribution of spontaneous plant species [ 31 ], few plant species co-occurred in all three decantation basins targeted in our study, among which were the two selected species P. tremuloides and E. elliptica . The soil contamination could also significantly reduce the number of species or modify the respective abundance of AMF species in a community [ 24 , 39 ]. Accordingly, of the 36 AMF OTUs detected overall in this study, only nine were found more than twice, with between one and to four OTUs only in each combination of plant species and contaminant concentration. Interestingly, despite the high level of petroleum hydrocarbon contamination (up to 91,000 µg TPH/kg of soil, which represents 9.1% (w/w) of TPH in soil), we detected AMF either in roots or in the plant rhizospheres that were obviously interacting with plants in this highly contaminated environment. Most of the sequences were formed by OTUs belonging to Glomerales (85.24%), and the remainder (less than 15%) consisted of OTUs from Diversisporales and Paraglomerales. These three orders were also previously reported from the roots of plants growing in high levels of metal contamination [ 44 ]. The confirmed occurrence of AMF under high contamination supports the idea that these fungi are effective partners in the detoxification processes and in the alleviation of abiotic stress in plants [ 8 , 15 , 16 , 17 , 24 , 25 , 40 ]. The ability of host plant species to strongly influence their AMF communities was previously documented [ 21 , 22 , 23 ]. However, the effect of petroleum hydrocarbon contamination on the selection of AMF taxa by plants is still poorly known. Here, the changes in AMF community structure were highly correlated with the host plant identity and the biotope (roots and rhizosphere soil). We found the influences of biotopes and hydrocarbon contamination on AMF community structure, with shift patterns differing among host plant species. We found that AMF communities associated with E. elliptica were not significantly affected by the concentration of petroleum hydrocarbons in both roots and rhizospheric soils ( Figure 3 ). This result did not support our hypotheses. Contrarily, the AMF communities associated with P. tremuloides were clearly affected by contamination concentration, which supports our first hypothesis. Interestingly, AMF community of roots were affected, while soil AMF community were not affected by petroleum hydrocarbon concentration. The finding was unexpected because the soil AMF community should be linked with the root AMF community in general, as AMF are obligatory plant root symbionts. There are three possible explanations for this observation. First, the AMF community of P. tremuloides roots were in transition phase when the sampling took place (cessation of oil refining activities by the petrochemical plant), assuming that soil AMF community might remain unchanged overtime during the season. Indeed, the AMF community associated with a host plant can change following the season or the growing stage of plants [ 26 ]. A second possible hypothesis is the close association between soil bacterial and fungal community and the AMF community that can increase the stability of the AMF community in soil. As suggested by the concept of plant holobiont, mycorrhizal networks of AMF serve as a backbone for the below-ground components of the holobiont [ 18 , 19 , 45 , 46 ]. There are various soil bacteria that have AMF hyphae as their ecological niche. Some of these bacteria can even form biofilm-like structures on the surface of hyphae [ 47 , 48 ]. These is still a lack of knowledge about how deep the intimate association between soil microbial communities and AMF could happen. However, considering the intimacy between AMF and interacting bacterial species, the resilience of AMF and soil microbial communities against environmental changes could come from both. Currently, one of the largest obstacles to applying AMF at contaminated sites is that the AMF community can be affected by the contamination [ 24 , 25 , 26 ], thus the expected effect from the induced shift of the AMF community by AMF inoculum can be mitigated by the contamination. This second scenario, if true, could be crucial to further developing strategies to remove contamination from soil using the AMF and AMF-interacting microbial community together instead of solely via AMF inoculation. This could be a means of stabilizing the functioning of inoculated AMF or bacterial species by stabilizing AMF and soil microbial community. Further efforts to screen and isolate bacterial species intimately interacting with AMF under high contamination, and to understand the interaction between those bacteria and AMF, should be made. The third putative scenario is the dispersion of AMF communities either by wind or by runoff, or even by birds, from the basin digs where the plants were growing [ 49 , 50 ]. At the same time, previous studies reported that host plants could favor certain AMF species to become dominant in their rhizosphere under particular environmental conditions [ 20 , 51 ]. Furthermore, evidence has been reported in support of a narrowed specificity of effective symbiotic partners due to the dialog response of both AMF and plants, which could exert selection pressure. Examples of selection have been known for a number of decades, such as the intimate AMF plant specificity reported between three legume species, Medicago sativa , Hedysarum coronarium and Onobrychis viciaefolia , and four Glomus species when grown in two soils with different phosphorus (P) availability [ 52 ]. In our study, OTU1 ( R. irregularis , VTX00114) was the most dominant taxon and represented 71% of all AMF sequences ( Figure 2 ). R. irregularis is one of the most common AMF species and is frequently found in diverse ecosystems. It was also reported as the dominant AMF species in various contaminated sites, such as soils contaminated with trace metals and petroleum hydrocarbons [ 24 , 25 , 26 ]. In our study, Claroideoglomus sp. (VTX00193) was the dominant OTU in the rhizosphere soil samples of P. tremuloides , except for the samples from the basin with the lowest contamination level, while R. irregularis was the dominant OTU in all samples from E. elliptica rhizosphere. At the same time, nearly all root samples, regardless of host plant species, did not show any difference in dominant AMF OTUs (except in the case of P. tremuloides from MC soil showing Aculospora sp. VTX00028 as the dominant species). Instead, they all shared R. irregularis as a common dominant AMF OTU. It has been known that AMF OTUs were highly associated with the three environmental factors (contaminant concentration, host plant identity, biotope) ( Figure 3 and Figure 4 ), which is in line with Velazquez, et al. [ 53 ], who reported that AMF occurrence under certain environmental conditions varied significantly between species. Not only the overall dominance of R. irregularis regardless of the contamination level, but the result of the db-RDA showing high level of association of R. irregularis with high contamination concentrations was also in line with previous reports suggesting that R. irregularis have a high tolerance in extreme environments. This species was also frequently found in sites contaminated with trace metals and known to mitigate the contamination effect on plants, which suggests the species as a promising partner for bioremediation of petroleum hydrocarbon contamination [ 24 , 26 ]. Recently, it was revealed from genome sequencing studies that the functional gene repertoire of different AMF species can vary significantly, implying a functional difference among different clades of AMF [ 27 , 28 ]. If a phylogenetic signal of AMF for their association with contamination can be found, it will suggest that certain common features might be associated with unique genes encoded in the genome of that clade that might be linked to their tolerance or stability against contamination stress. Surprisingly, we found that AMF species of the same phylogenetic clade have similar patterns of distribution against contamination concentrations and host plant identity. We found several OTUs belonging to the genus Diversispora ( Diversispora eburnea and Diversispora celeta ), Claroideoglomus ( Claroideoglomus sp. (VTX00193 and VTX00276) and Rhizophagus ( R. irregularis VTX00114 and Rhizophagus sp. VTX00113)). The db-RDA revealed that Diversispora and Claroideoglomus showed a tendency to be associated to the host plant P. tremuloides , but did not show unified patterns of distribution with contamination concentrations and biotopes. Thus, the conserved gene repertoire between these two genera is not likely to be related to AMF tolerance against petroleum hydrocarbon contamination. On the contrary, both OTUs (VTX00113 and VTX00114) that belong to the genus Rhizophagus showed clear correspondence with high contamination. Moreover, species from this genus have been continuously reported to be dominant under various polluted environments including extreme heavy metal contamination [ 24 , 26 ]. The results therefore suggest narrowing the target AMF taxon to the genus level for conducting a functional gene survey to understand the mechanisms of the tolerance against soil contamination. In summary, the high concentrations of petroleum hydrocarbon contamination considerably decreased the AMF diversity. This high contamination also greatly influenced the number of OTUs found in this study. However, AMF communities were not structured only by the level of petroleum hydrocarbon contamination. We showed that plant identity and biotopes also profoundly affected OTUs abundance and influenced their community structure. Overall, as reported in other studies of site contaminated with trace metals and petroleum hydrocarbons [ 24 , 26 ], R. irregularis was also found to be the dominant OTU in the three basins. Moreover, we found a strong association of OTUs of the genus Rhizophagus with high levels of contamination. The observed association in congruence with the accumulating reports of tolerance of Rhizophagus against abiotic contamination suggests the importance of Rhizophagus for future applications of AMF in bioremediation, as well as a survey of key genes for understanding AMF tolerance against trace elements and petroleum hydrocarbon stresses. The outcome of this investigation allowed us to trap and isolate two Rhizophagus irregularis strains which were deposited at the Canadian National Mycological Herbarium (DAOM), Ottawa, Canada under the accession numbers 242422 and 242423. The strain DAOM-242422 was used as an inoculant in a phytoremediation trial in the site of a former industrial landfill planted with willows [ 54 ]. Further investigations are required for better understanding the role of Rhizophagus spp. in anthropized environments."
} | 4,381 |
30296937 | PMC6174563 | pmc | 6,226 | {
"abstract": "Background Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated sugar source can be inhibitory to microbial growth and impair expression of the desired biosynthetic pathway. Results We utilized laboratory evolution to select Escherichia coli strains capable of robust growth in the presence of the IL, 1-ethyl-3-methyl-imidizolium acetate ([EMIM]OAc). Whole genome sequencing of the evolved strain identified a point mutation in an essential gene, cydC , which confers tolerance to two different classes of ILs at concentrations that are otherwise growth inhibitory. This mutation, cydC - D86G , fully restores the specific production of the bio-jet fuel candidate d -limonene, as well as the biogasoline and platform chemical isopentenol, in growth medium containing ILs. Similar amino acids at this position in cydC , such as cydC - D86V , also confer tolerance to [EMIM]OAc. We show that this [EMIM]OAc tolerance phenotype of cydC - D86G strains is independent of its wild-type function in activating the cytochrome bd-I respiratory complex. Using shotgun proteomics, we characterized the underlying differential cellular responses altered in this mutant. While wild-type E. coli cannot produce detectable amounts of either product in the presence of ILs at levels expected to be residual in sugars from pretreated biomass, the engineered cydC - D86G strains produce over 200 mg/L d -limonene and 350 mg/L isopentenol, which are among the highest reported titers in the presence of [EMIM]OAc. Conclusions The optimized strains in this study produce high titers of two candidate biofuels and bioproducts under IL stress. Both sets of production strains surpass production titers from other IL tolerant mutants in the literature. Our application of laboratory evolution identified a gain of function mutation in an essential gene, which is unusual in comparison to other published IL tolerant mutants. Electronic supplementary material The online version of this article (10.1186/s12934-018-1006-8) contains supplementary material, which is available to authorized users.",
"discussion": "Discussion ILs are a double-edged sword as a biomass pretreatment reagent: they are appealing because they can help efficiently extract sugar from recalcitrant lignocellulosic plant biomass, but often pose a detrimental effect on the downstream enzymes and microbial hosts for bioproduction of a target compound. In earlier reports, the improvement in IL tolerance has been shown to enhance product titers relative to strain productivity before engineering, but not at the levels possible under optimal growth conditions [ 14 , 16 , 46 , 47 ]. This inability to fully recover production levels after engineering strains for IL tolerance indicates that the IL impacts cell growth and biosynthetic pathways separately; the biosynthetic pathway was still affected, even though overall growth under these conditions was restored. In combination, even minor reductions in strain productivity would limit the development of one-pot processes or even the use of unwashed pretreated biomass. The E. coli cydC - D86G chassis is an important step forward in the field, as it is both IL tolerant and retains the optimal efficiency of producing advanced biofuels. Future processes could utilize this mutation in cydC to develop one-pot methods with [EMIM]OAc pretreated lignocellulosic biomass, without compromising biofuel production. We could not find the exact mechanism for the gain of function activity of the mutant cydC - D86G because cydC encodes an essential gene, a gene category that is challenging to utilize in metabolic engineering studies. Conditionally-essential gene collections are not yet widespread in bacteria, as has been completed for budding yeast [ 48 ]. The development of inducible CRISPRi libraries [ 49 ] will facilitate utilization of this gene reservoir and elucidate their cellular functions [ 50 ]. Spontaneous mutagenesis screens, such as via laboratory evolution, remain the standard for identifying advantageous gain-of-function mutations. As evidenced by the breadth of functional categories implicated by our proteomics data, ionic liquids have a pleiotropic impact on bacterial systems. Similar results have been reported in other model organisms: in the yeast S. cerevisiae , [EMIM]OAc has been reported to cause a growth impact that exceeds that of [EMIM]Cl or NaOAc [ 51 ], and is thought to deform cell wall structure and limit oxygen transfer from the culture media into cells [ 35 ]. While we did not detect a decrease of the expression of genes related to fatty acid synthesis from this analysis, we did detect a five-fold decrease in the abundance of an essential gene, plsB (see Additional file 3 : Table S2). PlsB is a critical for the downstream selection of fatty acids incorporated into membrane phospholipids, which could impact cell membrane structure or integrity [ 52 ]. However, the relationship between membrane lipid and protein composition and cydC - D86G remains to be unraveled. Previous studies have primarily resulted in the identification of native and non-native efflux pumps whose expression confers tolerance to [EMIM]OAc [ 14 , 16 , 53 , 54 ]. In this study, we identify a new mechanism for bacterial resistance to [EMIM]OAc that addresses both the IL toxicity impact on cell growth and also protects the production pathway from inhibitory impact. It is unclear why mutations in candidates from two different protein families, specifically, efflux pumps and CydCD complex both conferred tolerance towards IL. However, both categories are fairly ubiquitous across bacteria and have broad substrate specificity [ 55 ]. We have demonstrated that our mutant, cydC - D86G, is a gain-of-function mutant and the over-expression of wild-type cydC alone does not confer tolerance to this IL in E. coli . While the complete mechanism for this IL tolerance remains to be understood, we speculate that this point mutation renders CydCD-complexes more promiscuous or active, but retains the minimum activities required for cellular viability. The mutant allele of cydC could result in CydCD-complexes with additional capacity to enhance multi-genic functions in carbohydrate transport and protein turnover/post translational modifications, without necessarily exporting a toxic metabolite from the cytoplasm (see Fig. 6 ). These gross changes could result in a distinct cytosolic response, blocking both the downstream impacts [EMIM]OAc on cellular processes as well as protecting the cell from the burden imposed by the mevalonate pathway for limonene or isopentenol production. Fig. 6 CydC modulates many cellular activities. Proposed role of cydC and ionic liquid tolerance. Proteomic analysis and experimental validation in E. coli deletion strains implicate a suite of pathways involved in the cydC response to [EMIM]OAc. In the top panel , cells express a heterologous gene pathway to produce a desired product, turning sugar (blue hexagon) into the target compound (green hexagon). The addition of the IL [EMIM]OAc in the middle panel has a broad impact on cellular physiology, impacting respiration, inorganic ion response factors, transporters, which ultimately inhibit cell growth and desired production. However, the cydC - D86G mutant in the bottom panel has a distinct proteomic response, and instead upregulates unique gene function categories, such as [U] intracellular trafficking and secretion and [G] carbohydrate metabolism and transport (see Fig. 4 for complete legend), ultimately restoring both cell growth and desired biofuel production \n Our strategy to develop a microbial host chassis for the production of limonene in the presence of the IL, [EMIM]OAc, provides a template to obtain gain-of-function mutants applicable for cultivation under industrially-relevant conditions. By closely mimicking the conditions under which the target compound is produced, we obtained a single gain-of-function mutant in five independent trials in less than 40 h of incubation. In contrast to other laboratory evolution studies [ 15 , 56 , 57 ], we used an inhibitory concentration of the IL and included the potential burden of the limonene production plasmid in the screen. Future studies should consider the burden of a heterologous gene pathway as a fitness disadvantage when undertaking evolution studies. This study exemplifies the use of interdisciplinary approaches to classical genetics and microbiology in conjunction with rational strain engineering to provide an elegant blueprint for optimizing microbial biofuel production under otherwise unfavorable culturing conditions. Heterologous gene pathways are a metabolic burden and have idiosyncratic impacts on native microbial metabolism. While cydC - D86G strains were already competent to produce d -limonene, the production of isopentenol in the presence of exogenous [EMIM]OAc required the use of acetate deficient strains to achieve the highest titer of isopentenol under these stress conditions. In retrospect, it is clear that a heterologous gene pathway could impact the native host metabolism, as we observed when we initially sought to produce isopentenol under IL stress conditions. A unified strategy for engineering microbial hosts will be critical in the development of promising non-model microorganisms in one-pot processes when final product toxicity is inhibitory [ 58 , 59 ]. Metabolic flux models which incorporate data from all of these relevant impediments on central metabolism will advance our efforts to build robust cell factories."
} | 2,472 |
35921249 | PMC9804152 | pmc | 6,228 | {
"abstract": "Abstract Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH‐regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time‐consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H‐dependent redox reactions. The modular photo‐electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme‐ or substrate‐toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.",
"conclusion": "Conclusion In conclusion, many previously noted limitations of cyanobacteria used for cofactor regeneration can be overcome using MPS. For instance, the modularity allowed the extracellular regeneration of both reduced nicotinamide cofactors, NADH and NADPH, while cyanobacteria provide mainly intracellular NADPH. In nature transmembrane electron shuttling is described e.g. for the malate‐aspartate shuttle or the glycerol‐3‐phosphate shuttle,[ \n 58 \n , \n 59 \n ] thus the here presented concept represent a new‐to‐nature approach. Furthermore, the modular design allowed the fast evaluation of a panel of 14 different EREDs, without the requirement for the time‐consuming cloning of each individual enzyme into a cyanobacterial host. Additionally, key parameters, such as the concentration of the involved enzymes or the amount of cyanobacterial cells can easily be adapted, as demonstrated in the semi‐preparative ERED‐catalysed reduction of 2 a to ( R )‐ 2 b , reaching substrate concentrations of up to 50 mM and a volume of 50 mL. The approach also minimizes the risk of toxic effects of the heterologous enzyme on the host cells metabolism, as was previously reported for heterologous HicDHs in cyanobacteria. \n [24] \n Applying HicDHs with the presented system, allowed not only to overcome this toxicity but also to circumvent the reported problem of limited substrate uptake through the cell membrane. \n [24] \n The reaction rates and product formations increased as compared to previous approaches, which also allowed to handle toxic substrates such as imines. \n [25] \n \n However, the application of toxic substrates at higher concentration may be limited depending on the rate at which they are transformed to less harmful products. A further limitation of the MPS represents the required ADHs, which might also react with substrates that contain a carbonyl function, as experienced for substates 1 a and 12 a . When using EREDs a slow background activity was detected, which might turn out to be challenging in case the cyano‐enzyme display a different stereoselectivity than the enzymes applied in Module C. Furthermore, elongated reaction times with the ERED substrates resulted in reduced recoveries due to side reactions. A further challenge might be the application of the system at larger scale. While an upscaling to 50 mL was straight‐forward, even larger scales will have to address issues with self‐shading of the cells and reduced light penetration. Strategies to improve the performance of the presented systems include the testing of lower concentrations of NAD(P) + and optimizing the growth conditions of the cyanobacterium to accelerate photosynthesis. For example, the illumination conditions during growth are reported to have a significant influence on the photosynthetic activity. \n [23] \n Furthermore, engineering of the cyanobacterium itself, e.g., its NADPH utilization \n [22] \n or of the promotors to tune the heterologous expression levels, \n [24] \n have already been shown to be beneficial in related systems. The performance of the presented approach compared well with the established coupled enzyme regeneration system, using e.g. 2‐propanol for cofactor recycling, but has the advantage just to require light and water. The modular photo‐electron shuttling method was successfully demonstrated for 19 enzymes of four classes of redox reactions and for twelve different substrates. The modular photo‐electron shuttling (MPS) system presented herein leverages the photosynthesis‐driven cofactor recycling concepts based on cyanobacteria[ \n 21 \n , \n 22 \n , \n 23 \n , \n 24 \n , \n 25 \n , \n 26 \n , \n 27 \n , \n 28 \n , \n 29 \n , \n 30 \n ] to a new level and may open new avenues for light dependent biocatalytic redox reactions.",
"introduction": "Introduction Stereoselective redox reactions belong to the most important transformations in organic synthesis.[ \n 1 \n , \n 2 \n , \n 3 \n , \n 4 \n ] Typical examples are the asymmetric reductions of prochiral functional groups such as imines, ketones, or alkenes as well as stereoselective oxidations like C−H functionalization reactions or Baeyer–Villiger oxidations of selected molecules. Biocatalytic methods offer a broad spectrum of transformations for this portfolio of redox reactions,[ \n 5 \n , \n 6 \n , \n 7 \n , \n 8 \n ] as about one quarter of the known enzymes belong to the class of oxidoreductases.[ \n 9 \n , \n 10 \n ] The high stereoselectivity that biocatalysts usually exhibit is therefore mirrored by the broad application of biocatalytic oxidations and reductions in industry, especially in the synthesis of small molecule pharmaceuticals.[ \n 8 \n , \n 11 \n , \n 12 \n , \n 13 \n , \n 14 \n ] Most biocatalytic reductions require one equivalent of reduced nicotinamide cofactor (NADH or NADPH) as electron‐source. \n [1] \n In the case of monooxygenases, one oxygen atom of the co‐substrate molecular oxygen may for instance be incorporated into the product while the second atom is reduced to water, again at the expense of one equivalent of NAD(P)H. \n [2] \n Consequently, various methods for the regeneration of reduced nicotinamides have been established, using e.g. glucose, alcohols, formate, phosphite or even molecular hydrogen as sacrificial electron donor.[ \n 15 \n , \n 16 \n ] Alternative protocols apply chemical reductants, use light to enable the utilization of cheap donor molecules or directly employ electrons for the regeneration of the cofactor with electrochemical methods.[ \n 15 \n , \n 16 \n , \n 17 \n , \n 18 \n ] In general, many regeneration concepts show a poor atom economy, e.g. from glucose/2‐propanol only two hydrogen atoms are used. Besides the direct usage of electrons via electrodes for reductions (requiring additional redox mediators)[ \n 18 \n , \n 19 \n ] and maybe the use of molecular hydrogen, \n [20] \n arguably one ideal electron donor molecule is water, as it is ubiquitous in biocatalytic reactions and would lead to molecular oxygen as the only co‐product. This has been realized by coupling nature's photosynthesis with an intracellular enzymatic redox reaction (Figure 1 a). In the very first report, an NADPH‐dependent ene‐reductase (ERED) was heterologously expressed in Synechocystis sp. PCC 6803 (hereafter Synechocystis ).[ \n 21 \n , \n 22 \n ] The photosynthetic machinery of the cyanobacterial host liberated electrons from water, ultimately producing intracellular NADPH, which in turn was used by the recombinant ERED to perform the biocatalytic reduction of C=C‐bonds. Recently, this approach has been extended to other biocatalysts, including alcohol dehydrogenases (ADHs),[ \n 23 \n , \n 24 \n ] imine reductases (IREDs), \n [25] \n monooxygenases,[ \n 26 \n , \n 27 \n , \n 28 \n ] the AlkBGT hydroxylation‐system, \n [29] \n and carboxylic acid reductases. \n [30] \n \n Figure 1 Photosynthetic nicotinamide regeneration using cyanobacteria. a) Photosynthetic water oxidation provides NADPH for an heterologously expressed intracellular oxidoreductase (in the shown example an ene‐reductase, ERED). b) Intracellular photosynthetic NADPH production (Module A) coupled to an extracellular redox reaction (Module C) via electron shuttling. The photosynthetic water oxidation transfers the electrons via an intracellular ADH (Module A) to an alcohol/ketone shuttle (Shuttle) to an extracellular ADH for the regeneration of extracellular NAD(P)H (Module B) which is coupled to the extracellular NAD(P)H‐dependent oxidoreductase (Module C). EWG=electron withdrawing group. However, all of these systems report a range of shortcomings which prevent them from broad application and fast implementation, the most prominent being the challenges associated with cloning and expression of the target enzyme in the applied photoautotrophic organisms.[ \n 31 \n , \n 32 \n , \n 33 \n ] Especially the long period (months) required for the introduction of a target gene in a photoautotrophic host excludes a fast variation and investigation of different expression systems, promotors, constructs, and also a screening of enzyme panels or enzyme variants. Further issues include the problematic heterologous expression of enzymes that are toxic to the cells metabolism or a limited transfer of the substrate or product through the cell‐membrane of the living organism.[ \n 24 \n , \n 25 \n , \n 27 \n ] Finally, the photosynthetic machinery produces NADPH which limits the applicable enzymes to NADPH‐dependent ones.[ \n 21 \n , \n 22 \n , \n 24 \n ] Alternative systems that use isolated thylakoid membranes for cofactor recycling[ \n 34 \n , \n 35 \n ] as well as a whole‐cell system providing formate for extracellular NADH recycling have been reported. \n [36] \n Furthermore, light‐dependent regeneration of reduced nicotinamides has also been realized using organic photocatalysts, \n [37] \n and heterogeneous photocatalysts in combination with electron mediators.[ \n 38 \n , \n 39 \n , \n 40 \n , \n 41 \n , \n 42 \n ] However, such systems mostly rely on buffer components as electron source.",
"discussion": "Results and Discussion To overcome selected issues with cyanobacteria, like the challenge of heterologous enzyme expression and others, we designed a system where the electrons photo‐generated by the cyanobacterium are shuttled to the outside of the cell to drive there any redox reaction of interest. This would allow a modular set‐up and a simple exchange of the extracellular reaction (Figure 1 b). The modular approach would allow to use one single cyanobacterial strain to provide photosynthetic hydride equivalents in the form of both NADH and NADPH, for any extracellular redox reaction. In brief, in Module A an oxidized small shuttle molecule (e.g. ketone), that is permeable to the cell membrane, \n [43] \n is converted to its reduced form (e.g. alcohol) inside the cyanobacterium, thereby taking up one redox equivalent. After passing the cell membrane, the alcohol is re‐oxidized in Module B by an exchangeable external ADH, producing one equivalent of either NADH or NADPH, depending on the ADH‘s cofactor specificity. The reduced nicotinamide is then utilized in the third Module C, encompassing the exchangeable NAD(P)H‐dependent target redox enzyme. Overall, the reaction only requires one equivalent of water and light, and the only side product is molecular oxygen. To test the idea of modular photo‐electron shuttling (MPS, Figure 1 b), wild type Synechocystis sp. PCC 6803 ( Synechocystis wt) was initially chosen for Module A, as it was reported to possess native ADH activity on cyclic ketones. \n [26] \n For the back‐translation of the redox equivalents that were generated inside the cyanobacterium and transported through the cell membrane by the shuttle molecule into extracellular NADH, the NADH‐dependent alcohol dehydrogenase (ADH‐A) from Rhodococcus ruber DSM 44541[ \n 44 \n , \n 45 \n , \n 46 \n ] was selected as Module B. The ene‐reductase (ERED) OPR3 from Lycopersicon esculentum [ \n 47 \n , \n 48 \n ] was chosen as Module C, to reduce the C=C‐bond of 4‐ketoisophorone ( 1 a ) as test reaction (Table 1 ). Although OPR3 was reported to prefer NADPH, \n [49] \n it is known to accept NADH as well. \n [47] \n From several alcohol/ketone pairs investigated as shuttle molecules (Table S1), cyclohexanol/cyclohexanone was selected for the initial experiment combining all Modules (Modules A+B+C). Although the first experiment led to a conversion of just 4 % (Table 1 , entry 1 and Table S2), it indicated that the concept might work, since in the dark no trace of product was formed. The low conversion was attributed to the low native ADH activity in the wild‐type Synechocystis sp. PCC 6803. Consequently, the strain was substituted for a recombinant strain of Synechococcus elongatus PCC 7942, heterologously expressing the NADPH‐dependent ADH from Lactobacillus kefir ( Lk ADH).[ \n 23 \n , \n 50 \n ] In subsequent experiments the NADPH‐dependent Lk ADH was investigated also as catalyst for Module B as NADPH‐providing alternative to the NADH‐recycling ADH‐A. Furthermore, it turned out that acetone/2‐propanol worked even better as shuttle pair (Table S3). Employing now the recombinant Synechococcus elongatus (Module A) together with ADH‐A as cell free extract (Module B) allowed to reach 55 % conversion and 66 % using the purified enzyme (Table 1 , entries 2–3). Employing the Lk ADH (in purified form as well as CFE, with NADP + ) for NADPH recycling in Module B, allowed to reach quantitative conversion and excellent e.e.s (entries 4–5). Note that this translates to almost 10 turnovers of NADP + and two turnovers of the shuttle molecule for this first preliminary system.\n Table 1 ] 1 Proof of principle of the modular system exploiting photo‐electron shuttling (MPS) to provide reduced nicotinamide for the reduction of 1 a . \n \n \n \n \n Entry \n \n Module A \n \n Shuttle \n \n Module B \n \n Module C \n \n Light \n \n Dark \n \n conv. [%] \n \n e.e. [%] \n \n conv. [%] \n \n e.e. [%] \n \n 1 \n \n wt Syn . \n \n cyclohexanone (20 mM) \n \n ADH‐A (lyo.), NAD + \n \n \n OPR3 \n \n 4 \n \n n.d. \n \n n.c. \n \n n.d. \n \n 2 \n \n rec. S . elongatus \n \n \n acetone (5 mM) \n \n ADH‐A (purif.), NAD + \n \n \n OPR3 \n \n 66 \n \n 97 \n \n n.d. \n \n n.d. \n \n 3 \n \n rec. S . elongatus \n \n \n acetone (5 mM) \n \n ADH‐A (CFE), NAD + \n \n \n OPR3 \n \n 55 \n \n 96 \n \n 6 \n \n 89 \n \n 4 \n \n rec. S . elongatus \n \n \n acetone (5 mM) \n \n \n Lk ADH (purif.), NADP + \n \n \n OPR3 \n \n >99 \n \n 96 \n \n n.d. \n \n n.d. \n \n 5 \n \n rec. S . elongatus \n \n \n acetone (5 mM) \n \n \n Lk ADH (CFE), NADP + \n \n \n OPR3 \n \n >99 \n \n 95 \n \n 12 \n \n 92 \n \n 6 \n \n rec. S . elongatus \n \n \n acetone (5 mM) \n \n – \n \n – \n \n 32 \n \n 93 \n \n 10 \n \n 92 \n \n Module A : Synechocystis sp. PCC 6803 cells (wt Syn .; OD 750 =10) or cells of the recombinant Lk ADH‐ Synechococcus elongatus strain (rec. S. elongatus ; OD 750 =5). Module B : ADH‐A (CFE: 0.5 mg ml −1 or purified: 30 μL of a 2.06 mg mL −1 stock) and NAD + (1 mM), or Lk ADH (CFE: 0.5 mg ml −1 or purified: 3 μL of a 24.6 mg mL −1 stock) and NADP + (1 mM). Shuttle : As indicated. Module C : OPR3 (purified enzyme, 100 μg mL −1 ) and 1 a (10 mM). All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 1 mL). Reaction overnight (16 h) in a photoreactor with white light (430 μE m −2 s −1 ) at room temperature and 600 rpm. Samples in “dark” were covered with aluminum foil. c.=conversion; lyo.=lyophilized whole cells (2 mg mL −1 ); purif.=purified enzyme; CFE=lyophilized cell free extract. n.d.=not determined. n. c.=no conversion. Complete data set is shown in Tables S3 and S4. Product 1 b is prone to racemization. Wiley‐VCH GmbH The higher conversions might be explained by the known preference of OPR3 for NADPH. \n [49] \n Control reactions revealed a certain background activity of the cyanobacterium and low reactivity in the dark (Entry 6). This can be attributed to native ERED‐activity in the Synechococcus elongatus strain and possibly stored reducing equivalents in the cells. \n [51] \n For investigating the influence of essential parameters (cell density, concentrations of NAD(P) + and the shuttle, light intensity and reaction time) the substrate N ‐phenyl methylmaleimide ( 2 a ) was used (Figure 2 a), since this substrate is not prone to side reactions such as carbonyl reduction. The substrate 2 a was supplied to the reaction as a solution in DMSO (for the solvent tolerance of Synechococcus elongatus see Figure S2).\n Figure 2 Influence of various parameters on the reduction of 2 a in the MPS system. a) General reaction scheme for the reduction of 2 a to 2 b . b) Loading of recombinant Synechococcus elongatus cells (OD 750 =0–20). c) Variation of extracellular NADP + concentration (0–1 mM). d) Loading of the alcohol/ketone shuttle (provided as acetone, 0–20 mM). e) Influence of the illumination conditions (white light, 0–510 μE m −2 s −1 ). f) Control reactions, either without external Lk ADH, OPR3 or light, background reactivity of the recombinant Synechococcus elongatus and performance of the system with the wt Synechococcus elongatus (no internal Lk ADH). g) Comparison of the MPS with the background reactivity of the wt Synechococcus elongatus ; total recovery is the sum of recovered product and substrate. Module A : Recombinant Synechococcus elongatus cells (OD 750 =5, or as indicated), or wt. Synechococcus elongatus cells (OD 750 =5). Module B : Lk ADH (CFE, 0.25 mg mL −1 , 0.23 U 2‐propanol mL −1 ), NADP + (0.1 mM, or as indicated). Shuttle : Acetone (5 mM, or as indicated). Module C : OPR3 (purified enzyme, 100 μg mL −1 ), DMSO (4 % v/v), and 2 a (10 mM). All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 1 mL). Reaction overnight (16 h, or as indicated) in a photoreactor with white light (430 μE m −2 s −1 , or as indicated) at room temperature and 600 rpm. The experiments were performed in two biological replicates, each in triplicate. Error bars represent standard deviation. Samples in “dark” were covered with aluminum foil. c.=concentration. Increasing the amount of recombinant Synechococcus elongatus cells, measured as OD 750 , from 1 to 5 went in hand with an increase of product formation of ( R )‐ 2 b , reaching 93 % product formation at an OD 750 of 5 and quantitative product formation at an OD 750 of 10 (Figure 2 b). As the total recoveries were quantitative at an OD 750 of 5, this cell loading, corresponding to a dry cell weight of 1.2±0.1 mg mL −1 and chlorophyll a content of 34±2 μg mL −1 , was selected for future reactions. When varying the concentration of the nicotinamide cofactor NADP + , the reaction went almost to completion at a NADP + concentration as low as 0.1 mM, which corresponds to 96 turnovers for the cofactor (96 % conversion, Figure 2 c). A residual activity that was found when the external addition of NADP + was omitted, which was attributed to the cells native EREDs. A similar amount of product was detected in the absence of acetone due to the same reason. The conversion to the product increased to 89 % when the concentration of acetone was 2 mM (≈4.5 turnovers of acetone; Figure 2 d). However, due to the volatility of the shuttle molecule, 5 mM were supplied in the subsequent experiments. Following the time course of the system under varied illumination conditions revealed that increasing the light intensity to 510 μE m −2 s −1 photosynthetically active radiation (PAR), reduced the amount of produced 2 b . In fact, dimming the light intensity improved the system, as under illumination with 215 or 90 μE m −2 s −1 , the reaction was complete within 3 hours (Figure 2 e). The apparent specific activity of the overall MPS system [calculated after one hour and estimated by the formation of ( R )‐ 2 b ], increased from 68±10 U g CDW \n −1 to 79±10 U g CDW \n −1 when the light intensity was amplified from 90 to 215 μE m −2 s −1 but stayed then almost constant upon further increase of the light intensity. Therefore, an intensity of 215 μE m −2 s −1 was chosen as optimal for further experiments. These numbers compare well to the specific activities found for a recombinant Synechocystis strain expressing the ERED YqjM. \n [21] \n In this case, engineering of the NADPH supply in the strain was reported to almost double the specific activity of the recombinant YqjM. \n [22] \n Note that the light intensity used during cultivation was 80 μE m −2 s −1 , and adaptation of the cellular photosystem to these conditions is reflected in better performance of the MPS at such lower intensity (90 to 215 μE m −2 s −1 ). \n [23] \n To put these numbers into context, a PAR of 90 μE m −2 s −1 corresponds to the light intensity measured on a windowsill behind glass on an autumn day in Graz, Austria. Control reactions were performed omitting one of the integral components of the recycling system (Figure 2 f). About 26–28 % of ( R )‐ 2 b were formed within 16 hours if one of the external enzymes ( Lk ADH or OPR3) was omitted. When the wild‐type Synechococcus elongatus (lacking the recombinant Lk ADH) was used for Module A, 15 % of the product ( R )‐ 2 b were formed and when the recombinant Synechococcus elongatus was incubated with 2 a in the absence of all other components of the recycling system, 20 % of product were found. All these reactivities account for the background ene‐reductase activity of the Synechococcus elongatus . When the reaction was performed in the dark, about 10 % conversion to the product were found, originating from redox equivalents stored in the whole Synechococcus elongatus cells. The high product formations achieved using the optimized reaction conditions prove the necessity of every single component of the system. In agreement with reports about an ERED originating from Synechococcus elongatus , \n [51] \n the background reaction exhibited the same stereoselectivity as the target reaction (Figure 2 f), therefore the e.e. values of the product were not influenced by the background. For a better understanding of the background reactivity, the time course of the reactions using the MPS was compared with the wild type Synechococcus elongatus only (Figure 2 g). With the MPS system using OPR3, 61 % of 2 a were converted to 2 b within the first hour of the reaction, while only 4 % of 2 a were converted in the first hour by wild‐type Synechococcus elongatus . The slower rate of the background ene‐reduction shows that the contribution of the native ene‐reductases to the overall reaction is only minor when the recycling system is applied. In addition, going in hand with observations made earlier, the recovery of the background reaction was only 69 % after 24 hours, as the slowly transformed substrate could be consumed in side reactions, such as nucleophilic additions of free cysteine groups to the activated electrophilic β‐carbon of the substrate.[ \n 22 \n , \n 52 \n ] To explore the generality of the optimized MPS, a library of 14 different EREDs was tested in this system using the alkene reduction of 2 a as model reaction (Figure 3 a). As most EREDs are active with both reduced nicotinamide cofactors, NADH and NADPH, Lk ADH and NADP + were arbitrary chosen for Module B, except for the case of the more strictly NADH‐dependent Lac ER. For 12 out of 14 EREDs the product formation was quantitative or close to quantitative (i.e., 100 turnovers of NADP + ), demonstrating the broad applicability of the MPS system for ene‐reductases (Figure 3 b). Even for NerA from Rhizobium radiobacter , giving a product formation of 43 %, the MPS outperformed the (traditional) coupled enzyme regeneration system using Lk ADH and 2‐propanol as auxiliary substrate, which gave 4 % of product (experiment performed for comparison, Figure 3 b and Table S6). In the case of Lac ER, when ADH‐A was applied as Module B to recycle NADH, the traditional cofactor recycling (using ADH‐A and 2‐propanol) gave 86 % product, while MPS resulted in 47 % ( R )‐ 2 b (i.e., 47 turnovers of NAD + ; for the complete data set of both regeneration systems see Table S6).\n Figure 3 Applicability of the MPS for a library of EREDs and the reduction of various activated alkenes 1 a – 6 a . a) General reaction scheme for the reduction of 1 a – 6 a . b) Product formation of ( R )‐ 2 b testing a library of 14 EREDs for the reduction of 2 a with the MPS or the coupled enzyme regeneration system using LkADH and 2‐propanol (20 mM) as auxiliary substrate (with 2‐PrOH). For LacER, the Module B was exchanged for ADH‐A and NAD + . c) Panel of products obtained using various EREDs. Module A : Recombinant Synechococcus elongatus cells (OD 750 =5). Module B : Lk ADH (CFE, 0.25 mg mL −1 , 0.23 U 2‐propanol mL −1 ), NADP + (0.1 mM). In case LacER was used in Module C : ADH‐A (CFE, 0.25 mg mL −1 ), NAD + (0.1 mM). Shuttle : Acetone (5 mM). Module C : The indicated ERED (purified enzyme, 100 μg mL −1 ), DMSO (4 % v/v; for substrate 1 a no DMSO was used) and the indicated substrate 1 a – 6 a (10 mM). All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 1 mL). Reaction overnight (16 h; for substrate 1 a : 3 h) in a photoreactor with white light (215 μE m −2 s −1 ) at 21 °C and 600 rpm. The experiments were performed in at least two biological replicates. Error bars represent standard deviation. EWG=electron withdrawing group. For further data see Tables S6 and S7. Having shown that the MPS is applicable for a library of EREDs, the system was expanded to the reduction of other activated alkenes (Figure 3 c). Using the optimized conditions identified above, the initial test substrate 4‐ketoisophorone ( 1 a ) was quantitatively converted to its product ( R )‐ 1 b . The high e.e. of 97 % is noteworthy, as the chiral centre of the product is prone to racemization. \n [53] \n Additionally to the N ‐phenylated methylmaleimide ( 2 a ) also the non‐arylated derivative 3 a was transformed to the corresponding reduced products with absolute stereoselectivity (99 % e.e.) and >99 % conversion to the product. Employing the ERED YqjM for the reduction of a diacid, which are in general challenging substrates for EREDs, the MPS‐driven reduction of citraconic acid 4 a led to 38 % of the optically pure ( R )‐ 4 b (>99 % e.e). The system was further applied to less activated substrates such as the lactone 5 a and the coumarin 6 a , resulting in the formation of >99 % of 5 b and 27 % of the bulkier 6 b . The reactions were also compared to the substrate coupled recycling (using Lk ADH and 2‐propanol) and investigated in a variety of control reactions, e.g. testing the background reactivity of the recombinant Synechococcus elongatus cells, the reaction in the dark and in the absence of the ERED (see Table S7). For substrates 4 a and 6 a , the substrate coupled recycling gave higher product yields than the MPS (52 % for 4 a and 63 % for 6 a ). Analogous to substrate 2 a , the dark controls often gave around 10 % of product due to redox equivalents stored in the cells. In general, the background ERED activity of the cells was either too slow to be competitive or, in case of 4 – 6 a , was not detectable (Table S7). In case of 1 b , background reactions due to overreduction by Lk ADHs in Module A and Module B were overcome by stopping the reaction after 3 h. MPS reactions with substrate 6 a showed lower recovery compared to control reactions probably due to product or substrate depletion associated with photobleaching of Synechococcus elongatus . To be able to run the MPS system for the reduction of 2 a at a higher substrate concentration than 10 mM, it was tested at 20 mM at varied cell loadings (Table S5). At 20 mM substrate concentration and an OD 750 of 5 the amount of formed product ( R )‐ 2 b reached 13.6 mM (68 %). Doubling the cell loading to an OD 750 of 10 gave 91 % of the product (18.1 mM). Next, the reaction was scaled to 50 mL at a substrate concentration of 20 mM 2 a (188.7 mg) in a 250 mL Erlenmeyer flask using the ERED OPR3 (Figure 4 ). After incubation and illumination overnight, 95 % product formation was detected by GC, giving after column chromatography an isolated yield of 70 % enantiopure product ( R )‐ 2 b (133.4 mg). Motivated by this, the concentration of 2 a was tested also at 50 mM, which produced up to 46.8±1.1 mM product at an OD 750 of 25 (for details see Table S5). This translates to ten turnovers of the applied shuttle molecule acetone (5 mM) and 468 turnovers of NADP + .\n Figure 4 Upscaling of MPS for the reduction of 2 a . a) General reaction scheme for the reduction of 2 a to 2 b . b) Setup of the reaction. Module A : Recombinant Synechococcus elongatus cells (OD 750 =10) Module B : Lk ADH (CFE, 0.25 mg mL −1 , 0.23 U 2‐propanol mL −1 ), NADP + (0.1 mM). Shuttle : Acetone (20 mM). Module C : OPR3 (purified enzyme, 100 μg mL −1 ), no DMSO, and 2 a (20 mM). All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 50 mL). Reaction overnight (16 h, or as indicated) in a photoreactor using white light (300 μE m −2 s −1 , or as indicated) at room temperature and 140 rpm. Since the MPS system worked rather well for the various EREDs, we investigated whether also other classes of NAD(P)H‐dependent enzymes may be used instead of EREDs. For this purpose, the ERED from Module C before was exchanged for two enantiocomplementary keto acid dehydrogenases, namely the L‐HicDH from Lactobacillus confusus DSM 20196[ \n 54 \n , \n 55 \n ] and the D‐HicDH from Lactobacillus paracasei DSM 20008 (Figure 5 ).[ \n 55 \n , \n 56 \n ] Using 2‐oxo‐isocaproic acid ( 7 a ) as substrate allowed to reach the corresponding hydroxy acid ( S )‐ 7 b as well as the enantiomer ( R )‐ 7 b with 82–83 % product formation each in optically pure form (>99 % e.e.; up to 83 turnovers of NADP + ) (Figure 5 , Table S8). The aromatic substrate phenylpyruvate ( 8 a ) led to even higher product formation, reaching 88 % for the hydroxy acid ( S )‐ 8 b and 92 % for the product ( R )‐ 8 b (i.e., up to 92 turnovers of NADP + ). As both enantiocomplementary enzymes exhibit excellent stereoselectivities, both enantiomers of the products could be accessed with an e.e. of >99 %. Comparing the results obtained here with a classic enzyme coupled recycling system shows that the MPS leads to comparable amounts of product formation (Table S8).\n Figure 5 Combination of MPS with keto acid dehydrogenases. a) General reaction scheme for the reduction of 7 a and 8 a to both enantiomers of 7 b and 8 b . b) Products 7 b and 8 b , yields and e.e.s. Module A : Recombinant Synechococcus elongatus cells (OD 750 =5). Module B : Lk ADH (CFE, 0.25 mg mL −1 , 0.23 U 2‐propanol mL −1 ), NADP + (0.1 mM). Shuttle : Acetone (5 mM). Module C : L‐HicDH (CFE, 0.5 mg mL −1 , 1.8 U 8a mL −1 ) or D‐HicDH (CFE, 0.5 mg mL −1 , 4.7 U 8a mL −1 ), and the indicated substrate (10 mM, 7 a was added as stock solution in BG11, pH adjusted to 7.5; 8 a was added as stock solution in DMSO, final v/v 4 %) and the corresponding reactions were supplemented with 100 mM HEPES/NaOH pH 8. All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 1 mL). Reaction overnight (16 h) in a photoreactor with white light (215 μE m −2 s −1 ) at room temperature and 600 rpm. The experiment was performed in two biological replicates, each in triplicate. In a recent report, the two HicDHs were recombinantly expressed in a modified Synechocystis strain and applied for the biocatalytic reduction of α‐keto acids. \n [24] \n But due to low expression levels of the HicDHs in the Synechocystis strain, and limited substrate transport through the cell membrane, this system required high cell loadings (OD 750 of 20) and suffered from low reaction rates. Additionally, the whole‐cell catalysed reaction was demonstrated to be independent from light. In contrast, using the here presented MPS, substrate/product transport is no problem as the reaction happens outside the whole cell [additionally here the background reaction using only the recombinant Synechococcus elongatus cells or the dark reactions showed only low reactivity (Table S8)]. Similar to the HicDHs, IREDs have already been recombinantly expressed in Synechocystis sp. PCC 6803, \n [25] \n and although this whole cell catalyst allowed to convert up to 8 mM of imine substrate, the reduction reaction was not light‐dependent and relied on redox equivalents stored in the cells. Therefore, the reduction of substrates 9 a – 11 a was reinvestigated using the presented MPS system with the IRED A from Streptomyces sp. and IRED J from Kribbella flavida DSM 17836.[ \n 25 \n , \n 57 \n ] The MPS system allowed to reach a light dependent product formation for substrates 9 a – 11 a of up to 92 % (92 turnovers of NADP + , Figure 6 ).\n Figure 6 MPS combined with IREDs. a) General reaction scheme for the reduction of 9 a – 11 a . b) Products 9 b – 11 b , yields and e.e.s. Module A : Recombinant Synechococcus elongatus cells (OD 750 =5). Module B : Lk ADH (CFE, 0.25 mg mL −1 , 0.23 U 2‐propanol mL −1 ), NADP + (0.1 mM). Shuttle : Acetone (5 mM). Module C : The indicated IRED (CFE, 4 mg mL −1 ; IRED A, 0.4 U 9a mL −1 IRED J 1.16 U 9a mL −1 ), DMSO (5 % v/v), and the indicated substrate 9 a , 10 a or 11 a (10 mM). All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 1 mL). Reaction overnight (16 h) in a photoreactor with white light (215 μE m −2 s −1 ) at room temperature and 600 rpm. The experiment was performed in two biological replicates, each in triplicate. Again the results were mostly comparable with the results of the classic cofactor regeneration reactions reported in vitro (see also Table S9). \n [57] \n The employed IREDs gave the stereocomplementary products for the reduction of 10 a with an e.e. of 36 % for ( R )‐ 10 b (IRED A) and >99 % for ( S )‐ 10 b (IRED J). Interestingly, the formation of ( R )‐ 11 b , using either enzyme, was limited to about 50 % of the conversion that was obtained with the coupled enzyme recycling system (only Modules B+C, supplying an excess of 2‐propanol; IRED A: 70 % conversion; IRED J: 43 % conversion; Table S9). For all these imine substrates, very low background activity in the dark and no background imine‐reductase activity of the Synechococcus elongatus were detected. Furthermore, it is worth to mention that imine substrates are reported to be toxic for cyanobacterial cells and therefore, higher cell loadings were required for the in vivo system. \n [25] \n Herein, such toxicity was not an issue, possibly due to the enhanced reaction rates, leading to a fast decrease of the concentration of the potentially harmful substrate. Motivated by these results we went forward to use the MPS to fuel a Baeyer–Villiger oxidation catalysed by a monooxygenase (BVMO) with the required reduced nicotinamide cofactor. For this reaction using the MPS, only the substrate (cyclohexanone 12 a ) and oxygen are required as reagents (Figure 7 , for all details see Figure S3). All other components of the system are only present in catalytic amounts. In detail, besides the substrate, the monooxygenase requires one molecule of oxygen and NADPH to produce the product and one molecule of water. The water is utilized by the cyanobacterium to generate the required NADPH and half an equivalent of oxygen.\n Figure 7 MPS combined with monooxygenations. General reaction scheme for the Baeyer–Villiger monooxygenation of 12 a to 12 b . Module A : Recombinant Synechococcus elongatus cells (OD 750 =5). Module B : Lk ADH (CFE, 0.25 mg mL −1 , 0.23 U 2‐propanol mL −1 ), NADP + (0.1 mM). Shuttle : No shuttle molecule added as the intermediary 12 c acts as shuttle. Module C : CHMO (CFE, 2 mg mL −1 , 0.22 U 12a mL −1 ), and 12 a (10 mM). All in BG11 medium (with 5 mM HEPES/NaOH buffer, pH 8; supplemented with 1 mM MgCl 2 , final volume 1 mL). Reaction overnight (16 h) in a photoreactor with white light (215 μE m −2 s −1 ) at room temperature and 600 rpm. The experiment was performed in two biological replicates, each in triplicate. When the MPS/BVMO oxidation of 12 a (10 mM) was performed using acetone as shuttle as described above, 84 % of ϵ‐caprolactone ( 12 b ) as well as a small amount of cyclohexanol ( 12 c ) were formed within 16 h. Incubation of substrate 12 a only with the recombinant Synechococcus elongatus under illumination, thus in the absence of Modules B and C, resulted exclusively in the formation of the alcohol 12 c . The ketone reduction can be attributed to the activity of the recombinant Lk ADH in the cyanobacterium (compare Table S10 and Table S3). As this suggests that the substrate 12 a as well as the alcohol 12 c easily surpasses the cell membrane, it was envisioned that cyclohexanone/cyclohexanol may act as shuttle, thus acetone may be omitted. Indeed, the reaction in the absence of acetone as shuttle led to the formation of 85 % of 12 b (Figure 7 ). Following the reaction over time revealed that in a first phase the substrate 12 a is reduced to the alcohol 12 c which then is further converted to the product 12 b via oxidation (and thereby recycling the cofactor and substrate) and monooxygenation (Figure S3 and Figure S4). Note that the reduction of 12 a to 12 c has already been reported when heterologous BVMOs, expressed in Synechocystis sp. PCC 6803 were challenged with 12 a , in this case leading to a loss of product. \n [26] \n The problem was later overcome by using a faster BVMO. \n [28]"
} | 9,455 |
23534863 | PMC3712477 | pmc | 6,229 | {
"abstract": "Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.",
"conclusion": "Concluding remarks New high-throughput methods outperform earlier approaches in terms of resolution and magnitude and offer unprecedented insights into fungal community ecology. However, without awareness of methodological biases, limitations of markers or bioinformatics challenges, large-scale sequencing risks yielding artificial results and misleading conclusions. Thus, early claims of astonishingly high species richness in 454-sequenced amplicons were exaggerated, because of problems in distinguishing technical artefacts from true diversity. Although more sophisticated bioinformatics tools are now available, high-throughput assessment of species richness remains a major technical challenge. Furthermore, considering that even a species represented by a single spore would be recorded in a sufficiently deeply sequenced sample, the biological relevance of such assessments may be questioned. Absolute analyses of species presence and diversity are also sensitive to contaminations during sampling, laboratory processing and sequencing. We argue that the major benefit of high-throughput methods rather lies in the capacity to provide information about the main fungal colonizers in large numbers of samples, to a progressively decreasing cost in terms of money and laboratory labour. In the near future, automated processing of samples may increase the scope and statistical power of ecological studies even further. In addition, novel sequencing techniques continually increase data output, which in combination with rapidly expanding databases of entire genomes enables a development away from molecular markers and PCR amplification towards direct analysis of meta-genomes and meta-transcriptomes of complex fungal communities (Kuske & Lindahl, 2013 ).",
"introduction": "Introduction The increasing use of molecular markers to identify fungi and analyse fungal communities in a phylogenetic context has initiated a boom in fungal ecology and phylogenetics. Our understanding of the important roles of fungi in symbiotic and pathogenic interactions with plants, as well as in transformation of plant litter and nutrient cycling, is thereby rapidly increasing. In particular, high-throughput sequencing methods enable detailed, semiquantitative analysis of fungal communities in large sample sets and provide ecological information that extends far beyond that provided by previous methods in terms of detail and magnitude. The process from field samples to species abundance data involves a long series of steps, from sampling via laboratory handling to bioinformatics treatment ( Fig. 1 ). At each step, there is a risk of losing and distorting information. Here we present an overview of the steps involved, highlight potential pitfalls, discuss alternatives, and propose solutions. Fig. 1 Overview of the steps involved in high-throughput sequencing of fungal communities."
} | 1,024 |
28270586 | PMC5340875 | pmc | 6,231 | {
"abstract": "ABSTRACT Environmental cues can stimulate a variety of single-cell responses, as well as collective behaviors that emerge within a bacterial community. These responses require signal integration and transduction, which can occur on a variety of time scales and often involve feedback between processes, for example, between growth and motility. Here, we investigate the dynamics of responses of the phototactic, unicellular cyanobacterium Synechocystis sp. PCC6803 to complex light inputs that simulate the natural environments that cells typically encounter. We quantified single-cell motility characteristics in response to light of different wavelengths and intensities. We found that red and green light primarily affected motility bias rather than speed, while blue light inhibited motility altogether. When light signals were simultaneously presented from different directions, cells exhibited phototaxis along the vector sum of the light directions, indicating that cells can sense and combine multiple signals into an integrated motility response. Under a combination of antagonistic light signal regimes (phototaxis-promoting green light and phototaxis-inhibiting blue light), the ensuing bias was continuously tuned by competition between the wavelengths, and the community response was dependent on both bias and cell growth. The phototactic dynamics upon a rapid light shift revealed a wavelength dependence on the time scales of photoreceptor activation/deactivation. Thus, Synechocystis cells achieve exquisite integration of light inputs at the cellular scale through continuous tuning of motility, and the pattern of collective behavior depends on single-cell motility and population growth.",
"introduction": "INTRODUCTION Bacteria are able to sense and respond to multiple external cues in their local environment. These responses allow cells to move into optimal or away from suboptimal environments by using a variety of appendages, such as flagella or pili. The chemotactic networks responsible for transducing inputs into complex motility responses are among the most extensively studied of all biological systems ( 1 – 3 ). Much of the knowledge about signaling in bacteria comes from cellular responses to individual inputs, although in the environment cells must deal with combinations of dynamic and antagonistic cues. In photosynthetic microbes, light powers photosynthesis, but high light levels and UV irradiation can also be damaging to cells, and so such microbes have evolved multiple mechanisms to reduce damage and move into optimal light environments. One such behavioral response is phototaxis, by which cells detect a light source and move toward it (positive phototaxis) or away from it (negative phototaxis). This process has been extensively studied in the model unicellular cyanobacterium Synechocystis sp. PCC6803 (here, Synechocystis ) ( 1 , 4 – 8 ). Photosynthetic cells experience varied light intensities and wavelengths throughout the day, which depend on the local environment of the bacterium (e.g., soil versus water, or over the diel cycle). These wavelengths of light are differentially absorbed and used by the photosynthetic apparatus, which in turn affects the energetics and growth of the cell. Cells appear to sense the direction of light rather than a gradient of intensity ( 6 , 9 ), but we have limited understanding of how light is sensed, whether cells measure flux in a graded manner, how multiple signals are integrated, and how simple or complex light inputs are transduced to the bacterial motility machinery to achieve directional movement. In our Synechocystis strain (the Carnegie substrain; see Materials and Methods), phototaxis occurs through biased movement toward or away from an illumination source placed at an oblique angle to the surface (see Movie S1 in the supplemental material) ( 10 , 11 ). On a moist agarose surface, cells move toward a white light source, and over time communities of cells in finger-like projections emerge from an initially homogeneous distribution of cells ( 11 , 12 ). The extent of formation of the finger-like projections at a fixed time point after inoculation is a function of the initial inoculation density ( 12 ). Furthermore, light input is also an energy source that allows for cell growth and division, which impact collective motility behaviors over long time periods. Cells exhibit both spatial and population heterogeneity in their phototactic responses, and individual cells can respond within minutes to changes in light conditions ( 10 ). Movement directionality is determined only by the current light direction, rather than based on a long-term memory of previous conditions ( 10 ). Our previous measurements indicated that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions ( 10 ), similar to that seen with Pseudomonas aeruginosa chemotaxis ( 13 – 15 ). A recent study that used the Moscow substrain of Synechocystis sp. observed directed movement toward the light source rather than biased random walk behavior. This difference has also been observed at the population level, wherein an entire colony of the Moscow substrain moved toward white light; however, during phototaxis of the Carnegie substrain, a significant fraction of the cells remained in the space occupied by the original inoculation ( 16 ). The current study utilized only the Carnegie substrain. 10.1128/mBio.02330-16.1 MOVIE S1 Time-lapse imaging of Synechocystis cells illuminated by a single red LED. LED illumination is from the top of the movie (as for Fig. 1 and 3 ). Cells appear to move in biased random walks toward the light. One microliter of cells from a culture with an OD 730 of ~0.8 was placed in the center of a 50-mm plastic petri dish (BD Falcon) containing 0.4% (wt/vol) agarose in BG-11 medium. Upon adsorption of the inoculum onto the agarose, the plate was inverted and placed in front of a red LED (Roithner LaserTechnik GmbH; LED660N-03, 5 mm, 15 mW, 24° spread), such that the LED was positioned 50 mm from the center of the droplet (which had a diameter of 2.5 mm) at the level of the agarose. Imaging was conducted at the center of the droplet at 30°C, 24 h after being exposed to red light illumination. Download MOVIE S1, AVI file, 17.9 MB . Copyright © 2017 Chau et al. 2017 Chau et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . In Synechocystis , incident light activates photoreceptors ( 5 , 16 – 20 ), resulting in a signal transduction cascade that activates type IV pili ( 21 – 23 ), and motility is increased wherever cells have deposited extracellular polymeric substances (EPS) ( 11 , 12 ), suggesting that EPS provides a medium for cell-cell communication to drive collective behaviors. Simulations based on a minimal biophysical model demonstrated that the combination of a biased random walk with motility-enhancing EPS secretion was sufficient to generate communities with similar fingering spatial patterns and on similar time and length scales as those determined from experimental data ( 12 ). The phototactic response of cyanobacteria depends on the wavelength of the incident light. Light wavelengths ranging from red to green result in positive phototaxis, while cells do not move or exhibit negative phototaxis away from blue or UV light, which can damage DNA and other cellular components ( 1 , 7 , 8 ). Similar to plant phytochromes, some photoreceptors in Synechocystis exhibit two photoreversible conformational states upon absorption of light with a specific wavelength. TaxD1 (also known as PixJ1) is the major photoreceptor required for positive phototaxis. It is a cyanobacteriochrome that exhibits blue-green photoreversion and switches between a blue light-absorbing form ( P b ; λ max = 425 to 435 nm) and a green light-absorbing form ( P g ; λ max to 535 nm) ( 24 ). Some photoreceptor pairs also have overlapping absorption spectra (e.g., TaxD1 and PixD at 435 nm and TaxD1 and UirS at 535 nm), suggesting the potential for complementary or antagonistic roles in mediating positive and/or negative phototaxis ( 19 , 24 , 25 ). The molecular mechanism by which cells integrate a set of photoreceptor inputs is unknown. In this study, we examined Synechocystis phototaxis in response to light of different wavelengths and intensities and to multiple inputs with different incidence directions or wavelengths. We determined that at the single-cell level, green light and red light promote positive phototaxis by increasing motility bias toward the light source in an intensity-dependent manner, while blue light inhibits motility altogether. Synechocystis cells integrate multiple light inputs of the same wavelength, resulting in motility along the vector sum of the light sources and altered step sizes under illumination by competing light sources. Recovery of phototactic bias at the single-cell level after transient substitution of blue light for green light was much slower than the loss of bias when green light was removed, but this slow recovery could be suppressed by maintaining the green light input. Taken together, our results reveal how interactions among the components responsible for promoting or inhibiting phototaxis are transduced into a complex range of cellular and community behaviors.",
"discussion": "DISCUSSION Our study shows that phototactic bias in Synechocystis is intensity dependent under red and green light illumination, but the fraction of cells that are motile is independent of intensity, as is their speed in each direction, indicating that cells are primed for a change in light input that would require them to alter their motility bias. How do cells control this bias in an intensity-dependent manner? The maintenance of speed argues against a global increase in the pilus activity responsible for cellular locomotion ( Fig. 1D ; see also Fig. S2C in the supplemental material). Instead, our data suggest that pilus localization or activity control conserves the amount of pulling along the incidence direction; it is likely that the ratio of pulling toward versus away from the light dictates bias. A roughly equivalent fraction of pulling events must also occur perpendicular to the light, which may allow Synechocystis cells to tune their bias in response to changing conditions. Our data thus also predict that the fraction of photoreceptors that are active at any time is intensity dependent. Such behavior has been observed in the blue light flux-dependent activation of the PixD photoreceptor, through dissociation of the inactive PixD-PixE complex into its active components ( 27 ). The rapid changes in direction that we observed could be due directly to photoreceptor switching between active and inactive states, or to downstream components of the motility transduction pathway, such as competition between pili. We previously observed a similar maintenance of speed under dark conditions after switching from white light illumination ( 10 ), indicating that light is at least transiently not required for cellular motility. However, our observations in the current study revealed that speed determination is more complex; speed dropped somewhat when cells were switched from red light to dark conditions ( Fig. S5B ) and decreased more substantially when switched from green light to dark conditions ( Fig. S4A ). These differences may be explained by cellular energetics, whereby white light activates photosynthesis more effectively than single wavelengths and therefore cells have more energy. Speed did not drop to zero when switching from green to blue light ( Fig. S4B and C ), indicating that immediately previous exposure to green light is sufficient to transiently overcome the inhibitory effects of blue light on motility. Thus, our results with different wavelengths show the clear dependence of the motility response on specific photoreceptors, though it is difficult to decouple this response from the photosynthesis-derived energy production that also affects motility. Nevertheless, there is a long deactivation time scale after red/green illumination that is also evident in the reversal of bias after blue light inhibition ( Fig. 6C ). Single-cell tracking provides a powerful way to increase our understanding of the molecular pathways by which photoreceptors communicate with and activate type IV pili to mediate a specific phototactic response. For example, we determined that cells exposed to two perpendicular LEDs move in the direction of the vector sum of incidence directions ( Fig. 2B and 3 ). We envision two possibilities for achieving this behavior: (i) the input signals are combined before type IV pilus activation, or (ii) type IV pili pull in two different directions but cellular movement is a product of multiple pulling events, and the force vector that determines motion is along the vector sum. Our previous study ( 10 ) suggested that consecutive steps taken by a single cell over 1-s imaging intervals are often due to the retraction of a single pilus, arguing for the first hypothesis above, although if the two pili were synchronized, then our data would be inconclusive. Labeling pili for a direct readout of retraction, or accomplishing an indirect readout via traction force ( 28 ), would provide insight into this situation, particularly if combined with the determination of photoreceptor localization via use of fluorescent protein fusions. The ability of Synechocystis cells to sense light directionality is particularly striking, given that their size is on par with the wavelength of visible light. A recent study suggested that directional light sensing is possible in Synechocystis cells because they act as spherical microlenses. This allows cells to “see” a light source, because the light is focused on the edge of the cell opposite from the source. This focusing then triggers movement away from the focused spot, resulting in movement toward the light source ( 9 ). Our current study suggests that cells sensitively tune their response based on light intensity ( Fig. 1 and 4 ). Responses after exposure to opposing LEDs suggest that cells are able to detect intensity differences, polarizing their motion toward the brighter LED in a position-dependent fashion ( Fig. 2 and 3 ). When exposed to perpendicular LEDs, Synechocystis communities started moving in one direction and then turned 90° ( Fig. 2B ), indicating that the shape of the incident light cone determines the cellular response and that cells can adjust to new light directions rapidly, without residual motion in the original direction of movement. To the best of our knowledge, the current study is the first to address the integration of light directionalities and wavelengths by Synechocystis cells, revealing a broad range of behaviors that lead to regulation and competition among the photoreceptors that collectively contribute to the phototaxis response. The increase in speed when cells are illuminated by two opposing LEDs ( Fig. 3B ) means that the elimination of motility bias is not due to an inhibition of pilus retraction events. Speed only increased parallel to the light source ( Fig. 3B ), with larger steps in these directions ( Fig. 3D , panel ii), suggesting that exposure to the two LEDs altered the pulling activity along this axis. A previous study with Neisseria gonorrhoeae proposed a tug-of-war mechanism in which type IV pili drive persistent motility ( 29 ). While the details are likely different for Synechocystis , we hypothesize a similar model in which the increased cellular motion back and forth along the light axis creates mechanical tension within the cell envelope that upregulates some part of the process of pilus retraction, such as the frequency of extension/retraction and/or force generation. This model would require that the two opposing LEDs cause pulling in both directions simultaneously, in contrast to the single-LED situation. In water columns where freshwater Synechocystis is normally found, blue and green light are likely to be less absorbed than infrared or red light, and therefore blue and green light may be important light cues. The contrasting enhancement of phototaxis by green light and inhibition by blue light ( Fig. 4 ), coupled to the variable increase in proliferation caused by each ( Fig. 4 ), provides intriguing possibilities for their combined effects. We have provided the first evidence that blue light inhibits phototaxis by virtually inhibiting cell movement ( Fig. 4C ; Fig. S3 ). The intensity dependence of the competition between green and blue inputs ( Fig. 5 ) also suggests that none of the photoreceptors has a dominant effect. Nonetheless, reversion of motility bias from blue light occurs over a longer time scale ( Fig. 6C and D ) than that from other light inputs we measured, especially reversion from the dark ( Fig. 6B ). Blue light inactivates TaxD1, whose presence promotes positive phototaxis ( 5 , 8 ); however, blue light illumination does not have the same effect as deletion of TaxD1, since we did not observe any negative phototaxis ( Fig. 4C ). This difference likely occurs because blue light also turns on blue light-responsive photoreceptors, such as Cph2, PixD1, and UirS ( 7 , 16 , 19 , 26 ), suggesting that analysis of the motility of mutants (potentially multiple knockouts) under varied light conditions may enable further deconstruction of the blue light response in Synechocystis . Here, an important component of the combination of green and blue light was the degree of cell proliferation, which drove a switch from complete inhibition of motility at high intensity to an increase in phototaxis at intermediate intensity ( Fig. 5 ). Given that blue light appears to stimulate more proliferation and inhibit motility ( Fig. 4C ), it is intriguing to consider the possibility of negative feedback between proliferation and motility in Synechocystis , as has been observed in the swarming bacteria Pseudomonas aeruginosa ( 30 ) and Myxococcus xanthus ( 31 ). While there may be similar feedback in Synechocystis , such a stark switch between motility and proliferation cannot exist, as evidenced by the coupled increase in phototaxis and proliferation under green light illumination ( Fig. 4 ); perhaps motility is aided by proliferation due to the requirement for high EPS levels. Moreover, since speed is partially maintained when switching from green to blue light ( Fig. S4B and C ), the time scales of exposure to a particular wavelength must also matter and not just whether photoreceptors are activated; this effect may be due to the effects of green and blue light on proliferation. Most models of Synechocystis motility have neglected proliferation, as it is difficult to measure since it must be measured as a function of wavelength, on a surface, and as it varies in time and space. Microfluidics may be necessary to rapidly probe this parameter space, and quantitative analyses of community phenotypes could be used to infer the pattern of cell division and growth. Phototaxis represents both a signaling and a motility response, requiring consideration at the single-cell and community scales. Given that cyanobacteria routinely experience complex environments, it is critical to establish experimental systems, such as the one employed here, that enable tuning of the light input in terms of wavelength, flux, and directionality. The current study highlights the capacity of Synechocystis cells to integrate multiple inputs, rather than simply choosing to respond to a light signal that dominates other signals. This integration of signals likely occurs within and outside the cell through the interaction of signaling pathways with their photoreceptors and through competition among pili across the cell surface. Motility also depends on other factors, such as EPS concentration, which is determined by the local cell density ( 11 ); hence, motility also depends on the promotion of cellular proliferation by the light environment. Therefore, phototaxis on the community scale is a collective behavior that relies on a combination of signal transduction of a light input, surface sensing through EPS-mediated mobility enhancement, mechanical feedback on pilus activity, and the local and global effects of cell density. These connections can be generalized to other multicellular behaviors in which dynamic interactions among multiple factors over various time scales, length scales, and/or modes of action (chemical/mechanical/energetic) are critical for comprehending the emergent properties of the community."
} | 5,191 |
34370727 | PMC8407571 | pmc | 6,232 | {
"abstract": "Artificial neural networks, taking inspiration from biological neurons, have become an invaluable tool for machine learning applications. Recent studies have developed techniques to effectively tune the connectivity of sparsely-connected artificial neural networks, which have the potential to be more computationally efficient than their fully-connected counterparts and more closely resemble the architectures of biological systems. We here present a normalisation, based on the biophysical behaviour of neuronal dendrites receiving distributed synaptic inputs, that divides the weight of an artificial neuron’s afferent contacts by their number. We apply this dendritic normalisation to various sparsely-connected feedforward network architectures, as well as simple recurrent and self-organised networks with spatially extended units. The learning performance is significantly increased, providing an improvement over other widely-used normalisations in sparse networks. The results are two-fold, being both a practical advance in machine learning and an insight into how the structure of neuronal dendritic arbours may contribute to computation.",
"introduction": "Introduction Artificial neural networks have had a huge impact over the last couple of decades, enabling substantial advances in machine learning for fields such as image recognition [ 1 ], language translation [ 2 ], and medical diagnosis [ 3 ]. The inspiration for these tools comes from biological neuronal networks, where learning arises from changes in the strength of synaptic connections between neurons through a number of different plasticity mechanisms [ 4 – 7 ]. The development of artificial neural networks away from the limitations of biology has meant that state-of-the-art artificial intelligence algorithms differ fundamentally from the biological function of the brain. For example global backpropagation algorithms have access to information that may be unavailable to real synaptic connections [ 8 ] (but see also [ 9 ]). Nevertheless, a number of biophysical principles have been successfully reintroduced, using salient features of real neuronal networks to make advances in the field of artificial neural networks [ 10 – 17 ]. Here we show how the dendritic morphology of a neuron, which influences both its connectivity and excitability, produces an afferent weight normalisation that improves learning in such networks. Real neurons receive synaptic contacts across an extensively branched dendritic tree. Dendrites are leaky core conductors, where afferent currents propagate along dendritic cables whilst leaking across the cell membrane [ 18 ]. Larger dendrites increase the number of potential connections a cell can receive, meaning that more afferent currents can contribute to depolarisation [ 19 , 20 ]. Conversely, larger cells typically have lower input resistances, due to the increased spatial extent and membrane surface area, meaning that larger synaptic currents are necessary to induce the same voltage response and so bring a neuron to threshold [ 21 , 22 ]. It has recently been shown theoretically by Cuntz et al (2019) [ 23 ] that these two phenomena cancel each other exactly: the excitability of neurons receiving distributed excitatory synaptic inputs is largely invariant to changes in size and morphology. In addition, neurons possess several compensatory mechanisms to help maintain firing-rate homeostasis through both synaptic plasticity regulating inputs [ 24 , 25 ] and changes in membrane conductance regulating responses [ 26 , 27 ]. These results imply a consistent biophysical mechanism that contributes to stability in neuronal activity despite changes in scale and connectivity. We find that this mechanism is general and demonstrate it for artificial neural networks trained using backpropagation. The goal here is two-fold: firstly we produce results that outperform the current state-of-the-art for sparsely connected networks and secondly we demonstrate that learning is improved by dendrites in the ideal case where all gradient information is available to every synapse, as is the case with the traditional backpropagation algorithm. Changing connectivity has traditionally not played much of a role in feedforward artificial neural networks, which typically used fully-connected layers where each neuron can receive input from all cells in the preceding layer. Sparsely-connected layers have, however, long been used as alternatives in networks with a variety of different architectures [ 10 ]. Sparse connectivity more closely resembles the structure of real neuronal networks and a number of recent studies have demonstrated that larger, but sparsely-connected, layers can be more efficient than smaller fully-connected layers both in terms of total parameter numbers and training times [ 28 – 30 ]. The advantage in efficiency comes from the ability to entirely neglect synaptic connections that do not meaningfully contribute to the function of the network. The dendritic normalisation analysed in this paper has particular application here as it implies that relative synaptic plasticity will depend on the number of connections that a neuron receives, not necessarily their strengths. Sparse networks are also less likely to be overfitted to their training data as sparse representations of inputs are forced to focus on essential features of the signal instead of less-informative noise. To produce an appropriate sparse connectivity a number of regularisation techniques have been suggested; L 1 - and L 0 -regularisations [ 28 , 31 ] both penalise (the latter more explicitly) the number of connections between neurons during training. Mocanu et al (2018) [ 29 ], building on previous work [ 32 – 34 ], have recently introduced an evolutionary algorithm to reshape sparse connectivity, with weak connections being successively excised and randomly replaced. This procedure applies to both feedforward and recurrent artificial networks, causing feedforward networks to develop connectivities based on the properties of their inputs and recurrent networks to develop small-world and scale-free topologies similar to biological neuronal circuits [ 35 ]. Such networks have comparable performance to fully-connected layers, despite having many fewer parameters to optimise. Normalisation is another feature that has previously been shown to enhance learning in neural networks. In particular Ioffe & Szegedy (2015) [ 36 ] introduced batch normalisation, where the inputs over a given set of training data are normalised, and Salimans & Kingma (2016) [ 37 ] introduced L 2 -normalisation, where afferent synaptic weights are normalised by their total magnitude. The latter is reminiscent of heterosynaptic plasticity, where afferent synapses across a neuron depress in response to potentiation at one contact in order to maintain homeostasis [ 25 , 38 , 39 ]. Both techniques have been applied in fully-connected networks and both work to keep neuronal activities in the region where they are most sensitive to changes in inputs. Interestingly, existing studies of sparse networks do not typically include any normalisation. The normalisation that arises from the relationship between a real neuron’s morphology and connectivity provides a particularly powerful, and biologically realistic, way to normalise sparse inputs. Dividing the magnitude of individual synaptic weights by their number distributes activity across neurons whilst keeping each cell sensitive to changes in inputs; neurons therefore encode signals from the proportion of presynaptic partners that are active, providing a simple and broadly applicable technique to ensure faster convergence to optimal solutions.",
"discussion": "Discussion We have shown that the fact that excitatory synapses are typically located on dendrites produces an L 0 normalisation of synaptic inputs that improves the learning performance of sparse artificial neural networks with a variety of different structures. Such dendritic normalisation constrains the weights and expected inputs to be within relatively tight bands, potentially making better use of available neuronal resources. Neurons respond more to the proportion of their inputs that are active rather than the absolute number and highly-connected neurons are relatively less excitable. We believe that such a normalisation procedure is robust and should be applied to improve the performance of feedforward sparse networks. Other results on normalisation [ 36 , 37 ] have also demonstrated improvements in training performance in fully-connected feedforward networks. Such approaches work by keeping neurons relatively sensitive to changes in inputs and our results here can be seen as the sparse, and biologically justified, analogue, with similarly simple and broad applicability to the L 2 -normalisation introduced by Salimans & Kingma (2016) [ 37 ]. In situations of dynamic connectivity, the dendritic normalisation outperforms other techniques. The comparison between the heterosynaptic plasticity-like L 2 -normalisation and our dendritic L 0 -normalisation is particularly interesting. In real neurons the former relies on actively re-weighting afferent contacts [ 25 , 38 ] whereas the latter can arise purely from the passive properties of dendritic trees, and indeed would typically need the expenditure of additional energy to counteract. Neurons often display complementary functionality between passive structure and active processes, for example in the equalisation of somatic responses to synaptic inputs at different locations both dendritic diameter taper [ 50 ] and active signal enhancement [ 51 , 52 ] play a role. Synaptic normalisation is in a similar vein. The effects are, however, distinct in some ways: while both normalisations keep neurons sensitive to inputs, the responses to learning differ. Heterosynaptic plasticity enhances changes in the relative weighting of contacts, whereas dendritic normalisation increases the stability of well-connected neurons while allowing faster learning in poorly connected cells ( Eq 5 ). This makes dendritic normalisation particularly suited to situations with evolving connectivity. In the context of biological realism, the normalisation here has much room for development. We sought a straightforwardly demonstrable and quantifiable computational role for dendritic normalisation and so focussed on the most well-developed theories within artificial neural networks [ 53 ]. Such networks have a number of features that are impossible to implement in the brain, so more investigation into the benefits of size-invariant excitability in living systems is necessary. Firstly, a single artificial neuron can form connections that both excite and inhibit efferent cells, a phenomenon which is not seen in the connectivity of real neurons [ 54 ]. It is possible to regard the mix of excitatory and inhibitory connections as a functional abstraction of real connections mediated by intermediate inhibitory interneurons [ 55 ], but a more satisfying picture may emerge by considering distinct inhibitory populations of cells as we did in our self-organised network. Accordingly, it would be interesting to test the effects of implementing two distinct experimentally observed types of compartmental inhibition including somatic and dendritic inhibitory connections [ 56 , 57 ]. Secondly, we mostly train our networks using supervised backpropagation which employs global information typically unavailable to real synaptic connections. Whilst there is emerging evidence that biological networks are able to approximate the backpropagation algorithm in some circumstances [ 9 , 58 , 59 ], a variety of other learning algorithms are also regarded as biologically plausible models for training networks [ 6 , 7 , 12 , 16 , 17 ]. Such algorithms are another natural fit for our normalisation as it too is implemented biophysically through the morphology of the dendritic tree. Thirdly, the neurons here generally are rate-based with either saturating or non-saturating outputs. Spiking networks can have different properties [ 11 ] and spikes could be incorporated into any of the approaches described above. The final sections of the results, with sparse recurrent and self-organising networks, address some of these issues by showing the contribution of dendritic normalisation to different types of learning and showing its generality as a computational principle, but there are many more interesting avenues to explore. Dendrites in general have much more to offer in terms of artificial neural computations. Synaptic connections are distributed spatially over branched dendritic trees, allowing for a number of putative computational operations to occur within a single cell [ 60 ]. Dendrites are able to passively amplify signals selectively based on their timing and direction [ 18 ] and actively perform hierarchical computations over branches [ 61 , 62 ]. Cuntz et al (2019) [ 23 ] noted that while mean neuronal firing rates are size-independent, the timing of individual spikes is not necessarily unaffected by morphology [ 22 ]. This means that signals encoded by rates are normalised whereas those encoded by spike timing may not be, implying that the two streams of information across the same circuit pathways are differentially affected by changing connectivity. Dendrites additionally hold continuous variables through their membrane potential and conductances that shape ongoing signal integration [ 63 ]. Such properties have potential computational roles that, while sometimes intuitive, have yet to be systematically studied at the level of neuronal circuits. Overall, in line with the spirit of the emerging fruitful interaction between artificial intelligence and neuroscience [ 53 , 64 ], this study has two major consequences. The first is a practical normalisation procedure that significantly improves learning in sparse artificial neural networks trained using backpropagation. The procedure can be used effectively for any sparsely-connected layers in shallow or deep networks of any architecture. Given that sparse layers can display better scalability than fully-connected layers [ 29 ], we believe that this procedure could become standard in deep learning. Furthermore, the biological plausibility of the procedure means that it is highly appropriate as a component of more physiologically realistic learning rules. Secondly, we have taken the insights from Cuntz et al (2019) [ 23 ] and demonstrated a previously unappreciated way that the structure of dendrites, in particular their properties as leaky core conductors receiving distributed inputs, contributes to the computational function of neuronal circuits."
} | 3,687 |
27446046 | PMC4928248 | pmc | 6,233 | {
"abstract": "Lake Towuti is a tectonic basin, surrounded by ultramafic rocks. Lateritic soils form through weathering and deliver abundant iron (oxy)hydroxides but very little sulfate to the lake and its sediment. To characterize the sediment biogeochemistry, we collected cores at three sites with increasing water depth and decreasing bottom water oxygen concentrations. Microbial cell densities were highest at the shallow site—a feature we attribute to the availability of labile organic matter (OM) and the higher abundance of electron acceptors due to oxic bottom water conditions. At the two other sites, OM degradation and reduction processes below the oxycline led to partial electron acceptor depletion. Genetic information preserved in the sediment as extracellular DNA (eDNA) provided information on aerobic and anaerobic heterotrophs related to Nitrospirae , Chloroflexi , and Thermoplasmatales . These taxa apparently played a significant role in the degradation of sinking OM. However, eDNA concentrations rapidly decreased with core depth. Despite very low sulfate concentrations, sulfate-reducing bacteria were present and viable in sediments at all three sites, as confirmed by measurement of potential sulfate reduction rates. Microbial community fingerprinting supported the presence of taxa related to Deltaproteobacteria and Firmicutes with demonstrated capacity for iron and sulfate reduction. Concomitantly, sequences of Ruminococcaceae , Clostridiales , and Methanomicrobiales indicated potential for fermentative hydrogen and methane production. Such first insights into ferruginous sediments showed that microbial populations perform successive metabolisms related to sulfur, iron, and methane. In theory, iron reduction could reoxidize reduced sulfur compounds and desorb OM from iron minerals to allow remineralization to methane. Overall, we found that biogeochemical processes in the sediments can be linked to redox differences in the bottom waters of the three sites, like oxidant concentrations and the supply of labile OM. At the scale of the lacustrine record, our geomicrobiological study should provide a means to link the extant subsurface biosphere to past environments.",
"conclusion": "Conclusion Stratification of Lake Towuti’s water column gave rise to different biogeochemical conditions in deeper parts of the water column and at the water–sediment interface at the three study sites. Respiration processes led to the gradual depletion of electron acceptors with increasing water depth, with microbial Fe 3+ and SO 4 2- reduction occurring below the oxycline. As a result of microbial uptake of nitrogen and phosphorus, carbon burial increased with water depth while NH 4 + accumulated in anoxic bottom waters. Sediments at the shallow site exhibited more labile OM as well as higher pore water SO 4 2- concentrations and, consequently, harbored the highest cell densities and potential SRR. Retrieved eDNA sequences confirmed the role of microbial degradation within the water column, with some aerobic and anaerobic heterotrophic elements potentially linked to the epilimnion and hypolimnion. Nevertheless, eDNA was substantially degraded in the uppermost sediment layers at all three sites, leading to the gradual loss of genetic information. Fingerprinting of iDNA revealed taxa common in SRB consortia, along with known iron reducers and methanogens at all sites. Our results attest that Lake Towuti’s sediments support microorganisms displaying complementary metabolic capabilities related to sulfur, iron and methane cycling. Relatively high SRR could be maintained in these ferruginous sediments through a cryptic sulfur cycle driven by iron reduction. The related loss of the sedimentary OM sorption capacity over time would then promote OM remineralization to methane. However, ferric iron phases may persist in deeper sediment layers, questioning the availability of organic substrates rather than that of reactive iron. To conclude whether they be related to climate or in-lake processes, redox changes in the water column appear to lead to variable burial of OM, electron acceptors and reactive metal species in the sediments. Regarding the entire lacustrine record, a long-term shift to more oxic conditions would lead to persistence of electron acceptors in deeper sediments and could promote metabolic activity by the subsurface biosphere.",
"introduction": "Introduction Lake Towuti is a tropical 200 m deep tectonic lake seated in ophiolitic rocks and surrounded by lateritic soils ( Lehmusluoto et al., 1995 ; Russell and Bijaksana, 2012 ). It is part of the Malili Lakes system, comprising several interconnected lakes, including Lake Matano, the 10th deepest lake in the world (~600 m). Its location on Sulawesi, Indonesia ( Figure 1A ) renders Lake Towuti’s sediments prime recorders of paleoclimatic changes in the tropical Western Pacific warm pool ( Russell et al., 2014 ). The tropical climate and the lateritic weathering of the (ultra)mafic catchment of the Malili Lakes system ( Figure 1B ) cause a strong flux of iron to the lake. Surrounding lateritic soils are typically related to limonite types, with mostly goethite (α-FeOOH) and ferrihydrite (Fe 2 O 3 ⋅0.5H 2 O) transported to the basin ( Crowe et al., 2004 ; Golightly, 2010 ) as well as some hematite (Fe 2 O 3 ) and detrital magnetite (Fe 3 O 4 ). High iron fluxes to the lake may exert a decisive constraint on bioavailable phosphorus in the epilimnion as it is scavenged by iron (hydr)oxides, likely driving Lake Towuti’s water column toward severely nutrient-limited conditions. However, anoxia in stratified water column can lead to iron reduction and partial release of adsorbed P into the water at the oxycline and below ( Zegeye et al., 2012 ). Biogeochemical and microbiological data gathered from nearby Lake Matano reveal persistent anoxia in the deeper part of Lake Matano’s water column ( Crowe et al., 2008b ; Jones et al., 2011 ) with organic matter (OM) degradation through methanogenesis ( Katsev et al., 2010 ; Crowe et al., 2011 ). Although Lake Towuti is anoxic at greater depths as well, it is less deep and can mix periodically ( Haffner et al., 2001 ), presumably causing transient bottom water oxygenation ( Costa et al., 2015 ). FIGURE 1 Lake Towuti location and settings. (A) Map of Asia and Oceania displaying the location of Sulawesi Island. (B) Map of Sulawesi illustrating the geological context of the Malili lake system (modified after Calvert and Hall , 2007 ). (C) Bathymetric map of Lake Matano and Lake Towuti (modified after Herder et al., 2006 ) displaying the three sites at which gravity cores were retrieved. Sites 1–3 correspond respectively to water depths of 153, 200, and 60 m, with oxygenation conditions at the water–sediment interface decreasing with water depth. Once buried, ferruginous sediments likely support microbial communities, which can utilize a range of metalliferous substrates ( Crowe et al., 2007 ). Although microbial activity decreases dramatically below the water–sediment interface and with increasing sediment depth ( Kallmeyer et al., 2012 ), even this low activity can have an appreciable impact on both sediment composition and biogeochemical cycles over long-time periods ( Berner, 1980 ; Freudenthal et al., 2001 ; Horsfield and Kieft, 2007 ). In addition, iron minerals are also suspected to strongly adsorb DNA ( Cecchio et al., 2005 ; He et al., 2008 ). Upon cell lysis, nucleic acids are released into the surrounding water and sediment and partitioned between sorption to reactive Fe surfaces ( Pietramellara et al., 2008 ; Ceccherini et al., 2009 ) and uptake or degradation via microbial metabolisms ( Corinaldesi et al., 2007 , 2008 ). Binding to metal oxides and colloids ( Ceccherini et al., 2009 ; Cleaves et al., 2011 ) could result in preservation and persistence of extracellular DNA (eDNA) in the lacustrine record ( Pietramellara et al., 2008 ), providing a valuable archive of genetic information ( Corinaldesi et al., 2011 ). However, since metal-reducing bacteria have the capacity to solubilize structural Fe and utilize adsorbed nutrients ( Dong et al., 2003 ; Crowe et al., 2007 ), the sediment-bound eDNA should not be totally recalcitrant ( Baldwin, 2013 and references therein) and could serve as a labile organic substrate for sedimentary microbes ( Corinaldesi et al., 2007 ). Its concentrations should then depend on the complex interplay between these processes ( Dell’Anno and Corinaldesi, 2004 ). Altogether, Lake Towuti provides the opportunity to examine microbial populations in an iron-dominated and sulfate-poor ecosystem with dynamic redox conditions and infer sorption and diagenetic processes arising from subsurface microbial activity. In order to investigate the relationship between biogeo chemistry and microbial processes in these iron-rich anoxic sediments, we retrieved short sediment cores from Lake Towuti in 2013 and 2014 at three different depths ( Figure 1C ). The sediment was analyzed for pore water geochemistry, total cell counts and potential sulfate reduction rates (SRR) as well as eDNA and intracellular DNA (iDNA). These data provide a background for geomicrobiological and biogeochemical analysis of the long (>100 m) drill cores that were retrieved during the ICDP (International Continental Scientific Drilling Program) Towuti drilling campaign in spring/summer 2015.",
"discussion": "Discussion Water Column Conditions and Organic Matter Sedimentation Varying oxygenation levels in the water column, especially at the water–sediment interface are expected to lead to differences in production, degradation, and burial of OM in sediments at the three sites ( Katsev and Crowe, 2015 and references therein). Chlorophyll a concentrations are highest around 50 m water depth, while transmission profiles indicate suspended matter close to the oxycline at 130 m water depth ( Figure 2 ), pointing toward a redox interface similar to the one observed in the water column of nearby Lake Matano, although less steep ( Crowe et al., 2008a ). At the intermediate site, a long-term shift in the oxycline was recorded during the mid-Holocene in the form of elevated ferric iron and siderite (i.e., FeCO 3 ) concentrations ( Costa et al., 2015 ). Due to the low sedimentation rates (i.e., 2 mm year -1 ; Vogel et al., 2015 ), redox fluctuations would be hard to detect at our sampling resolution. TOC contents indicate increased OM burial from the shallow to the deep site ( Figure 3A ), associated with a shift toward more recalcitrant OM composition (i.e., higher C org /N ratio). Although degradation processes in an anoxic water column are generally slower ( Katsev and Crowe, 2015 ), iron reduction below the oxycline can foster desorption of labile OM and microbial uptake of nitrogen and phosphorus ( Zegeye et al., 2012 ), thereby increasing carbon burial linked to bacterial production. Remineralization of organic compounds leads to the production of NH 4 + , which can be oxidized or accumulate, depending on the presence of suitable reactants in the water column. NH 4 + concentrations in bottom waters ( Table 1 ) indicate that degradation of sinking particulate OM is indeed continuous in the water column, leading to the accumulation of less reactive organic material at the deep site ( Amon and Benner, 1996 ). This interpretation is also consistent with the diverse taxa identified from our eDNA sequences, which corresponded to both aerobic and anaerobic heterotrophs, namely Actinobacteria, Nitrospirae, Chloroflexi, and Thermoplasmatales, and secondarily Alpha- and Gammaproteobacteria ( Figure 4 ; and Supplementary Material). However, final discrimination of eDNA sources cannot be achieved. For example, Actinobacteria could originate from soils eroded from the catchment, transported to the lake and preserved within refractory OM. Although we did not detect any photoautotrophs, certain elements can still be assigned to planktonic assemblages coming from the epilimnion (e.g., Nitrospirales) and hypolimnion (e.g., Anaerolineales). Our interpretation is that labile OM from primary producers is rapidly degraded by heterotrophic species that are active in the water column, resulting in the preferential preservation of heterotrophic over phototrophic sequences in the eDNA ( Vuillemin et al., 2016 ). This is also consistent with the transmission decrease at the oxycline (i.e., 130 m), which we interpret as iron reduction leading to desorption of phosphorus compounds and thereby promoting anaerobic heterotrophic processes. Data from Lake Matano show that dissolution of particulate Fe 3+ occurs at and below the pycnocline along with the liberation of phosphorus and concomitant production of methane and ammonia ( Crowe et al., 2008b ; Katsev et al., 2010 ). Pore Water Geochemistry High Ca 2+ and Mg 2+ concentrations in pore waters ( Figure 3A ) likely reflect continued weathering of mafic/ultramafic minerals derived from the catchment of the Malili Lake system ( Kadarusman et al., 2004 ). Although fairly constant, variations in these profiles could possibly imply precipitation and/or dissolution of authigenic carbonates (i.e., Ca–Mg–FeCO 3 ) within the sediment. At the intermediate and deep site, the presence of authigenic carbonates can be inferred from peaks in TC profiles. The diagenetic sequence known for freshwater sediments in relation to pore water geochemistry (i.e., Ca–Mg siderite, calcite, ankerite, dolomite) suggests the formation of early diagenetic siderite ( Berner, 1980 ; Matsumoto and Iijima, 1981 ), which is consistent with the decline in dissolved Fe 2+ concentrations at the deep site. Lake Matano and Lake Towuti are both iron-rich but also extremely low-sulfate environments, far lower than most aquatic environments studied to date ( Crowe et al., 2014 ). Moreover, microbial reduction processes in the water column likely take place at or directly below the oxycline, as shown for SO 4 2- concentrations in Lake Matano, which decrease drastically along with a rapid increase of dissolved Fe 2+ ( Crowe et al., 2008a ). In Lake Towuti, SO 4 2- concentrations measured at the water/sediment interface decreased from the shallow to the deep site ( Table 1 ), supporting our assumption that sulfate reduction already takes place in the anoxic bottom water of the intermediate and deep site. As a result pore water SO 4 2- concentrations in the uppermost sediment samples decreased toward the deep site and were rapidly depleted with sediment depth at all sites. Pore water Fe 2+ concentrations show the opposite trend, increasing from the shallow to the deep site and with core depth, showing an increasingly steeper gradient between sites with maximum values around 45 μM ( Figure 3A ). The production, consumption, and dynamics of NH 4 + in bottom waters seemed to differ between the three sites as indicated by their respective concentrations (i.e., <6, 20, and 13 μM) and the very similar NH 4 + concentrations in the uppermost sediment sample at all three sites (ca. 25 μM). The NH 4 + profile of the deep site is indeed the only one that clearly indicates NH 4 + diffusion out of the sediment into the bottom waters. Whereas nitrification could take place in the oxic bottom waters of the shallow site, the potential accumulation of NH 4 + in the anoxic bottom waters of the two other sites remains unclear. Sediment Microorganisms and Extracellular DNA Remineralization Several studies showed that bacterial abundance and diversity in lake sediments correlate with environmental parameters such as salinity, pH, OM content, and sediment depth ( Nam et al., 2008 ; Zeng et al., 2009 ; Borsodi et al., 2012 ), and can also reflect climatic variations due to forcing of conditions during sediment deposition ( Dong et al., 2010 ; Vuillemin et al., 2013 , 2014 ). Lake Towuti’s surface sediments are characterized by comparatively high cell densities that decrease from the shallow to the deep site. At all sites, cell counts decrease by one order of magnitude over the uppermost 5 cm below the water–sediment interface and remain more or less constant over the rest of the core ( Figure 3B ). The corresponding iDNA concentrations follow a similar trend, whereas Shannon indices show more variability between the three sites. Potential SRR measurements with radiotracer ( Figure 3A ) further demonstrate that SRB are present and viable at all sites, but are more active at the shallow site, showing some correspondence to Shannon indices. We interpret these data as the positive response of bacterial populations to geochemical conditions corresponding with higher bottom water SO 4 2- concentrations and increased burial of labile OM and reactive ferric iron into the sediment. With regard to recording of past lake conditions by eDNA, water temperatures of Lake Towuti are approximately 28°C throughout the year, which is rather unfavorable for eDNA preservation ( Lindahl, 1993 ; Renshaw et al., 2015 ). Although sorption capacities in the sediment were expected to vary between the three sampling sites, eDNA distribution patterns are similar, displaying a rapid decrease in the uppermost 5 cm followed by constant but low concentrations over the rest of the cores ( Figure 3B ). The decline of eDNA Shannon indices also indicates a gradual loss of genetic information associated with the degradation and shortening of eDNA sequences over time ( Corinaldesi et al., 2008 ). Such decrease as a function of sediment depth can be attributed to a combination of sediment sorption capacity, microbial uptake, and degradation as well as rates of cell lysis. We argue that the observed lower eDNA concentrations result from an overall decrease in metabolic activity and cell lysis rate, along with the immediate degradation of the free eDNA fraction resulting in diminishing Shannon values. In the long-term, eDNA preservation would greatly depend on metabolic turnover rates and its potential stabilization by ferric mineral phases ( Glasauer et al., 2003 ; Pinchuk et al., 2008 ). In this context, preliminary results indicate that reactive ferric iron persists down to 15 m sediment depth at concentrations up to nearly 2 wt% ( Simister et al., 2016 ). Phylogeny of Intracellular DNA and Presumed Metabolic Processes Microbial fingerprinting ( Figure 4 ; and Supplementary Material) revealed the presence of microorganisms related to taxa commonly known for iron and sulfate reduction as well as methanogenesis, indicating that the autochthonous microbial communities have the metabolic capacity for these processes. Among Proteobacteria, certain taxa were affiliated with Denitratisoma ( Fahrbach et al., 2006 ), Thiobacillus ( Schedel and Trüper, 1980 ; Haaijer et al., 2006 ), and Acidiferrobacter ( Hallberg et al., 2011 ), indicating putative capacity for both lithotrophic and organotrophic processes driven through use of reduced sulfur and iron as electron donors. However, these Beta- and Gammaproteobacteria typically exhibit metabolic versatility with the capacity to shift between different modes of facultative metabolisms ( Masters and Madigan, 1983 ; Hiraishi and Hoshino, 1984 ; Ferguson et al., 1987 ), making it impossible to assess metabolism from taxonomic information alone. This could also indicate that these taxa are planktonic in the bottom waters, then benthic at the water–sediment interface until burial. Interestingly, sequences of Deltaproteobacteria at the shallow and intermediate site included taxa assigned to the candidate order Sva0485 and Desulfovibrio , which are often reported as part of SRB consortia ( Kleindienst et al., 2014 ; Bar-Or et al., 2015 ). These sequences were correspondingly identified in sediments with the highest measured SRR. In addition, detection of Desulfuromonas M20-Pitesti, Deferrisoma and Geobacter -related sequences may indicate that the metabolic capacity to reduce sulfur and iron is evenly distributed across all three sites ( Slobodkina et al., 2012 ; Greene, 2014 ). DGGE fragments affiliated with Thermincola also point at a metabolic potential for iron reduction ( Zavarzina et al., 2007 ). In addition, taxa related to Ruminococcaceae and Clostridiales were representative of heterotrophic anaerobes that can produce fermentative hydrogen via ferredoxin (i.e., Fe 2 S 2 protein cluster) reduction ( Hallenbeck, 2009 ). Their concomitance with Methanomicrobiales suggests that H 2 /CO 2 reduction is a likely pathway for methane production ( Thauer et al., 2008 ; Kaster et al., 2009 ), although formate and alcohols could also be used ( Ohren, 2014 ). Together, our measurements of potential sulfate reduction and our findings of microbial taxa commonly involved in microbial sulfate and iron reduction suggest that ferruginous sediments support microorganisms that degrade OM via sulfate reduction, in spite of the extremely low sulfate concentrations, in collaboration with diverse iron-reducing bacteria, fermenters, and methanogens. Seemingly, such complementary metabolisms imply desorption of OM following iron reduction and its remineralization to methane. The low pore water SO 4 2- concentrations (single μM) and the relatively high potential SRR (single to tens of nmol cm -3 day -1 ) also demonstrate that the sulfate pool is turned over within days. This suggests that reoxidation of reduced sulfur compounds occurs through a cryptic S-cycle driven by iron ( Norði et al., 2013 ; Hansel et al., 2015 ) in order to maintain these high SRR. A likely mechanism for such recycling is the disproportionation of elemental sulfur linked to ferric iron reduction ( Thamdrup et al., 1993 ; Holmkvist et al., 2011 ). Moreover, unless methanogens are being outcompeted by iron reducers ( Roden and Wetzel, 1996 ), the persistence of ferric iron in deeper sediment could be seen as an indicator for processes involved in anaerobic oxidation of methane ( Hallam et al., 2004 )."
} | 5,503 |
37636112 | PMC10448772 | pmc | 6,235 | {
"abstract": "Under agroforestry practices, inter-specific facilitation between tree rows and cultivated alleys occurs when plants increase the growth of their neighbors especially under nutrient limitation. Owing to a coarse root architecture limiting soil inorganic phosphate (Pi) uptake, walnut trees ( Juglans spp.) exhibit dependency on soil-borne symbiotic arbuscular mycorrhizal fungi that extend extra-radical hyphae beyond the root Pi depletion zone. To investigate the benefits of mycorrhizal walnuts in alley cropping, we experimentally simulated an agroforestry system in which walnut rootstocks RX1 ( J. regia x J. microcarpa ) were connected or not by a common mycelial network (CMN) to maize plants grown under two contrasting Pi levels. Mycorrhizal colonization parameters showed that the inoculum reservoir formed by inoculated walnut donor saplings allowed the mycorrhization of maize recipient roots. Relative to non-mycorrhizal plants and whatever the Pi supply, CMN enabled walnut saplings to access maize Pi fertilization residues according to significant increases in biomass, stem diameter, and expression of JrPHT1;1 and JrPHT1;2 , two mycorrhiza-inducible phosphate transporter candidates here identified by phylogenic inference of orthologs. In the lowest Pi supply, stem height, leaf Pi concentration, and biomass of RX1 were significantly higher than in non-mycorrhizal controls, showing that mycorrhizal connections between walnut and maize roots alleviated Pi deficiency in the mycorrhizal RX1 donor plant. Under Pi limitation, maize recipient plants also benefited from mycorrhization relative to controls, as inferred from larger stem diameter and height, biomass, leaf number, N content, and Pi concentration. Mycorrhization-induced Pi uptake generated a higher carbon cost for donor walnut plants than for maize plants by increasing walnut plant photosynthesis to provide the AM fungus with carbon assimilate. Here, we show that CMN alleviates Pi deficiency in co-cultivated walnut and maize plants, and may therefore contribute to limit the use of chemical P fertilizers in agroforestry systems.",
"conclusion": "5 Conclusion In agreement with our working hypotheses, this study provides evidence that (1) the inoculum reservoir formed by walnut roots allows the mycorrhization of maize roots through the formation of a CMN, thereby giving access to maize fertilization residues; (2) hyphal connections under shortage of P result in a benefit comparable to the P-sufficient condition without a CMN; and (3) because of a slower root growth rate than maize, walnut invests more carbon in the development of the AM fungus than maize does. These findings support the idea that in an agroforestry system, the presence of perennial root systems enables the maintenance of an active AM fungal network, which constitutes an AM reservoir allowing root colonization of the annual crop, and authorizing low phosphorus fertilization input. CMN formation is a facilitative mechanisms between trees and companion crops favorable to their growth and development ( Ingleby et al., 2007 ; Bainard et al., 2011 ; Bainard et al., 2012 ). These results are also in accordance with the stress-gradient hypothesis that predicts that low-fertilized agricultural lands would benefit more from intercropping than high-fertilized lands ( Brooker and Callaghan, 1998 ; Zhu et al., 2023 ). Therefore, mycorrhization of tree saplings with a suitable AM fungus able to develop CMN favorable to alley annual crop may result in a benefit comparable to P fertilization and would therefore be a recommendable agronomical practice in the framework of the agroecological transition of agricultural systems.",
"introduction": "1 Introduction Inorganic phosphate (Pi) availability, as the orthophosphate anion PO 4 \n − , is a major factor constraining plant growth and metabolism in many soils worldwide ( Bates and Lynch, 2001 ). Consequently, large amounts of Pi fertilizers are used to ensure plant productivity in most conventional agricultural systems ( Smith et al., 2011 ). However, only approximately 10%–30% of the P fertilizer is taken up by the roots, with a substantial part accumulated in the soil as residual P not readily available for plants ( Syers et al., 2008 ). In addition, injudicious and untimely application of chemical fertilizers in agricultural field has generated environmental concerns, including soil degradation and water eutrophication ( Tilman, 2001 ; Foley, 2005 ; Conley et al., 2009 ). To mitigate environmental damages, it is crucial to enhance P-use efficiency in crop production through the diminution of the application of P fertilizers and utilization of residual P and other P pools from soils ( Xue et al., 2016 ). As a substitute to conventional cropping systems, which rely on large inputs of chemical fertilizers to sustain production, agroforestry is a low-input design that combines trees with annual crops in various combinations or sequences ( Nair, 1993 ). Agroforestry systems aim at reducing the need for inputs through minimizing losses and maximizing internal cycling of nutrients ( Smith, 2010 ). One of the major arguments in favor of agroforestry is the fact that a mixed system of trees and annual crops uses natural resources such as light, water, and nutrients more efficiently than a monoculture. As resources are used more efficiently, less chemical fertilizers are required to the benefit of the overall environment ( Postma, 2005 ). A fundamental aspect of agroforestry is to favor inter-specific facilitation between tree rows and cultivated alleys, where plants increase the growth and survival of their neighbors ( Callaway, 1998 ), particularly regarding limited resources such as nutrients ( Schoeneberger et al., 2012 ; Battie-Laclau et al., 2020 ). In the maintenance of soil P fertility under alley cropping systems, the role of roots is at least as important as that of aboveground biomass ( van Noordwijk et al., 2015 ). Belowground facilitation occurs when one species makes previously unavailable P available to the other, due to the exudation of organic acids, phosphatases, or rhizosphere acidification resulting in increased P availability ( Hinsinger et al., 2011 ). Since a decade ago, numerous studies have also pointed to the importance of soil-borne arbuscular mycorrhizal (AM) fungi of the phylum Glomeromycota ( Tedersoo et al., 2020 ) in mediating inter-plant facilitation processes ( van der Heijden et al., 2008 ; Montesinos-Navarro et al., 2016 ). In soils with strong Pi-fixing capacity, or where Pi is limiting, plant demand for this nutrient exceeds the rate at which it diffuses into the root zone, resulting in zones of Pi depletion surrounding the roots ( Smith and Read, 1997 ). AM fungi help to overcome this limitation by extending their extra-radical hyphae from root surfaces to areas beyond the P depletion zone ( Hayman, 1983 ; Javot et al., 2007 ). When roots are colonized by AM fungi, soil Pi is acquired at the soil–fungus interface through high-affinity fungal phosphate transporters (PHTs) located in the extra-radical mycelium ( Rui et al., 2022 ). In the plant interfacial apoplast of fungal arbuscules, Pi is acquired by plant PHT located on the peri-arbuscular membrane ( Harrison et al., 2002 ; Javot et al., 2007 ). The Pi uptake pathway of mycorrhizae may dominate Pi uptake in AM symbiosis, which is heavily dependent on AM-induced PHT1 members ( Casieri et al., 2013 ; Rui et al., 2022 ). Consequently, AM fungi have the potential to maintain crop yields at low soil P levels through a more effective exploitation of available P sources. It has been estimated that inoculation with AM fungi might reach a reduction of approximately 80% of the recommended fertilizer P rates under certain conditions ( Jakobsen et al., 1992 ). In return, AM fungi are supplied with sugars and lipids from the host plant ( Gutjahr and Parniske, 2013 ; Roth and Paszkowski, 2017 ). Conversely, the importance of the role of mycorrhizal symbiosis in providing Pi to the plant usually declines when soil phosphate concentration is elevated ( Kiers et al., 2011 ). Many trees species used in tropical and temperate alley cropping systems such as ash, cocoa, coffee, eucalyptus, olive tree, paulownia, poplar, rubber ( Cardoso, 2002 ; Janos, 2007 ; de Kroon et al., 2012 ), and walnut (the common name given to the species of deciduous trees belonging to the genus Juglans ) exhibit a high dependency on symbiotic AM fungi for their development. Coarse roots and relatively few roots per plant limit soil Pi uptake ( Brundrett, 1991 ; Comas and Eissenstat, 2009 ; Comas et al., 2014 ). By providing wood, ornament, and nutrition value to human beings, and food and a habitat to wildlife, walnuts belong to the most important trees in the northern hemisphere, ecologically and economically ( Bernard et al., 2018 ). Because of its short growing season, sparse canopy, and deep rooting system, walnut is an ideal species for agroforestry and belongs to the dominant trees used in temperate alley cropping ( Wolz and DeLucia, 2018 ). Intercropping of Juglans spp. is aimed at increasing growth and quality of walnut trees together with providing an early financial return to help offset the costs associated with establishing walnut orchards ( van Sambeek and Garrett, 2004 ; Mohni et al., 2009 ; Wolz and DeLucia, 2019 ). Before walnut plantations reach economic maturity, intercrops—winter cereals ( Triticum spp.), alfafa ( Medicago sativa ), soybean ( Glycine max ), or summer crops (e.g., maize)—are the only source of income during the first 5 to 10 years; thereafter, both trees and intercrops produce simultaneously ( Mary et al., 1998 ). Among these AM-dependent intercrops, maize ( Zea mays L.) is one of the most cultivated crops for both staple food and industrial usage, in tropical and temperate soils worldwide, with phosphorus being a major growth-limiting factor for commercial maize production ( Brady and Weil, 2008 ). The role of AM fungi in mixed plant communities is applicable due to their low host specificity: extra-radical hyphae can connect the roots of different plant species to form a common mycelial network (CMN) ( Ingleby et al., 2007 ). This inter-plant mycorrhizal connection promotes facilitation through differential access to the nutrient pool from the common hyphal network and plant-to-plant nutrient transfers ( Fitter et al., 1998 ; Martins and Cruz, 1998 ; Leake et al., 2004 ; Simard and Durall, 2004 ; Hauggaard-Nielsen and Jensen, 2005 ; Ingleby et al., 2007 ; van der Heijden and Horton, 2009 ; de Carvalho et al., 2010 ; Walder et al., 2012 ; Fellbaum et al., 2014 ; Gorzelak et al., 2015 ; Montesinos-Navarro et al., 2016 ). Focusing on the effect of intercrops on tree growth, trees display a larger growth than that observed in simple forestry systems, due to a better mineral nutrition even in low input systems ( Jose et al., 2000 ; Rivest et al., 2010 ). To date, improved tree growth has been ascribed to the likely ability of tree roots to intercept and uptake a significant proportion of nutrient fertilizer residues from the crop rooting zone ( Smith, 2010 ), but the contribution of inter-plant mycorrhizal connections to this process remains poorly investigated. Because improving tree mineral nutrition through agroforestry systems is a low-cost means to increase tree growth and to shorten the development duration before tree harvesting ( Chifflot et al., 2009 ), this information is of importance to assess the overall costs or benefits of growing trees and annuals. In this study, we investigated the extent to which intercropping with maize contributes to the mineral nutrition and biomass production of young hybrid walnut plants through the formation of a common AM fungal mycelial network. To this aim, we experimentally simulated an agroforestry system using a homemade experimental device in which young walnut hybrid rootstocks were connected or not by a common mycelial network to maize plants grown under contrasting Pi supply levels. This experimental device was made of two separate compartments in which walnut and maize were planted and connected by a third compartment in which the extra-radical mycelium responsible for mineral nutrient supply for the plants was separated by fine nylon nets from the associated host roots. Two months after plant sowing, we analyzed plant growth parameters, nutrient contents, and expression of plant genes coding mycorrhiza-inducible PHT1 transporters, here identified by phylogenic inference of orthologs. We hypothesized that (1) the inoculum reservoir formed by walnut roots would allow the mycorrhization of maize roots through the formation of a CMN, thereby giving access to maize fertilization residues; (2) hyphal connections under P-deficient conditions would result in a benefit comparable to P-sufficient conditions without a CMN; and (3) due to a slower growth rate than maize, walnut would invest more carbon in the development of the mycorrhizal fungus than maize.",
"discussion": "4 Discussion 4.1 The inoculum reservoir formed by walnut roots allows the mycorrhization of maize through the formation of a CMN that gives access to maize fertilization residues Irrespective of the Pi supplied to Z. mays recipient plants, non-inoculated maize roots connected to mycorrhizal walnut rootstocks displayed F%, M%, and A% values reaching 100%, 30%, and 25%, respectively. These results demonstrated that whatever the Pi fertilization regime applied to maize, the inoculum reservoir formed by AM walnut roots enables the mycorrhization of maize recipient roots through the development of a common mycelial network (CMN). Consistently, extra-radical hyphae emanating from walnut root were visualized in the two Pi treatments after trypan blue staining of the nylon mesh and substrate contained in the fungal pipes close to Z. mays roots. This observation showed that the extra-radical mycelium that developed outside AM walnut roots have crossed the 40-µm nylon mesh to reach the recipient compartment and further colonize maize roots. Consistently, the high-affinity phosphate transporter RiPT1 of R. irregularis and the maize phosphate transporter ZmPHT1;6 were only expressed in maize connected to mycorrhizal RX1 donor plants. There was also evidence that AM walnut plants connected to recipient roots have access to maize Pi fertilization residues via the CMN as shown by higher stem diameter, shoot, and root dry biomasses recorded in mycorrhizal RX1 saplings as compared to non-inoculated walnuts in both Pi treatments. This finding demonstrated that walnut developing AM symbiosis gains growth benefit from the Pi supplied to maize through hyphal connections with walnut sapling. In addition, AM walnuts connected to recipient plants happened to be responsive to the extent of maize fertilization, as inferred from significantly lower M% and A% in the P condition relative to the P/10 treatment. This showed that Pi shortage in the maize compartment sustains walnut mycorrhization, while a 10-fold higher Pi fertilization restricts the intra-radical development of R. irregularis in RX1. Earlier studies have reported a significant suppression of AM symbiosis upon high Pi supplies ( Rausch et al., 2001 ; Breuillin et al., 2010 ; Bonneau et al., 2013 ; Kobae et al., 2016 ). When plants consume more Pi to provide photosynthetically fixed carbon than that furnished by mycorrhizal hyphae, the host limits soil Pi uptake through the mycorrhizal pathway by reducing carbon allocation to the symbiont in order to save energy ( Treseder and Allen, 2002 ; Blanke et al., 2005 ; Gavito et al., 2019 ). In agreement with this line, a decreased root carbon content, along with an increased leaf soluble Pi concentration and effective photochemical quantum yield of PS II in AM walnuts compared to NM plants, was only significant in the Pi-limiting condition. In the process of phosphate suppression of mycorrhization, Pi acts systemically to repress the expression of symbiotic genes, including phosphate transporters ( Breuillin et al., 2010 ), resulting in lower AMF colonization ( Salmeron-Santiago et al., 2021 ). However, in the present study, the transcript abundance of JrPHT1;1 and JrPHT1;2 in the roots of RX1 was similar irrespective of the Pi supply to maize plants. Because the cumulative Pi amount acquired through the mycorrhizal pathway and its translation into biomass yield did not differ in AM walnut saplings between the two Pi fertilization regimes, it is likely possible that the expression of mycorrhiza-inducible PHT1 transporters measured after symbiosis establishment may not actually reflect the Pi flux at the symbiotic interface ( Grace et al., 2009 ; Walder et al., 2015 ; Sawers et al., 2017 ; Watts-Williams et al., 2019 ). In this respect, it has been proposed that the regulation of the abundance of Pi transporters in the periarbuscular membrane is probably more dependent on post-transcriptional and post-translational regulation events ( Grace et al., 2009 ; Smith et al., 2011 ; Walder et al., 2015 ). As previously observed in maize plants ( Tian et al., 2013 ; Polcyn et al., 2019 ; Liang et al., 2022 ), increasing Pi fertilization did not significantly decrease mycorrhizal colonization, suggesting that phosphate inhibition of internal mycorrhization may not be a general paradigm, depending on host and fungal genotypes ( Peña Venegas et al., 2021 ). 4.2 Common arbuscular mycorrhizal network alleviates Pi shortage in walnut and maize plants Relative to a 1.3 mM Pi supply, a 0.13 mM Pi fertilization of non-mycorrhizal maize plants led to a significant reduction in stem height and diameter, leaf number, Pi and N content, and shoot and root biomass. As previously observed ( Almagrabi and Abdelmoneim, 2012 ; Ma et al., 2021 ), this result showed that maize growth is limited by phosphate deficiency. In NM maize exposed to P-sufficient treatments relative to Pi-limiting conditions, higher leaf count, dry mass, plant height, and stem thickness have also been reported: as an indicator of crop growth and development, when N is amply supplied, plant height is known to increase as P availability increases, similar to stem thickness ( Coetzee et al., 2016 ). In contrast to what was observed in NM controls, in AM maize plants cultivated under the P/10 supply, all growth and nutritional parameters rose to a level similar to that recorded for NM plants under the P-sufficient treatment, indicating that mycorrhization of maize plants alleviated Pi deficiency under the P/10 supply. These responses to mycorrhization in low P soil are consistent with previous studies demonstrating that maize crop benefits from mycorrhizal associations ( Calderón-Vázquez et al., 2011 ; Ma et al., 2021 ). They also fit with the “optimal trade principle” predicting that plant growth will most strongly benefit from symbiosis with AM fungi under conditions of low soil phosphorus supply ( Johnson, 2010 ; Johnson et al., 2015 ). We concluded that under maize Pi shortage, the mycorrhizal connection between walnut and maize roots alleviates Pi deficiency in the recipient plant through a better mineral nutrition. This resulted in maize growth and nutritional benefits comparable to the P-sufficient condition without a common arbuscular mycorrhizal network. As expected from the absence of mycorrhiza connections between plants when walnut donor roots were not inoculated with R. irregularis , the Pi fertilization regime of maize had no impact on the parameters measured in NM walnut plants. This result supported the absence of solute diffusion through the air gap. With regard to walnut growth benefits gained from the Pi supplied to maize through hyphal connections relative to non-fertilized NM walnut plants, we observed that a 0.13 mM Pi fertilization led to a significant increase in root collar, stem height, leaf Pi content, and shoot and root DW. This underlines that under maize low Pi fertilization, the mycorrhizal connection between walnut and maize roots alleviates Pi deficiency in the donor plant through a better mineral nutrition. Contrary to maize, mycorrhizal growth benefits of walnut plants in terms of root collar and dry mass were conserved under high Pi fertilization of maize, even though intra-radical colonization was reduced. Altogether, these results support the idea that walnut is more dependent on the Pi uptake mycorrhizal pathway than maize to sustain its development owing to a coarse root architecture that has a limited intrinsic ability to absorb soil nutrients ( Bates and Lynch, 2001 ). We concluded that a common arbuscular mycorrhizal network is able to overcome phosphate shortage in walnut and maize plants. 4.3 \n R. irregularis acts as a stronger carbon sink in walnut than in maize When comparing mycorrhizal walnut and maize plants grown under the P/10 supply relative to NM plants, a significant decreased root carbon content along with an increased effective photochemical quantum yield of PS II, was only observed in RX1, while both plants displayed a higher leaf Pi concentration. With respect to the S/R biomass ratio as a measure of the carbon cost for AM establishment ( Chen et al., 2010 ), mycorrhizal colonization decreased the shoot to root biomass partitioning only in RX1 grown under the P/10 supply. These findings indicated that AM fungal colonization enhances walnut investment in root biomass development. Taken together, these results showed that mycorrhization-induced Pi uptake generates a higher carbon cost for walnut donor plants than for maize plants by increasing walnut plant photosynthesis to feed the AM fungus with carbon assimilate. In support of this rationale, when using walnut trees ( J. nigra L., C3-plant) and maize (C4-plant), which display distinctly different ratios of 13 C/ 12 C (∂ 13 C), analysis of ∂ 13 C from an AM mycelium taken from the surrounding environment of intercropped walnut and maize roots indicated the transfer of walnut photosynthates to the AM mycelium ( van Tuinen et al., 2020 ). With regard to the potential mechanisms underlying a larger carbon investment in AM symbiosis of walnut than maize, Graham et al. (1997) proposed that a high mycorrhizal dependency, as displayed by walnut, may reflect a greater availability of the carbon supplied by the host. On the opposite, because plants with rapid growth rate need more carbon and absorb more soil nutrients to achieve fast growth ( Lovelock, 2008 ), less photosynthates may thus be available to feed the CMN. According to Elser et al. (2000) , plant growth rate is usually negatively related to C/N content ratio. In the current study, when compared to NM plants, mycorrhization of maize with R. irregularis through the CMN led to a significant decrease in leaf C/N ratio under the maize Pi-limited condition, but not in the Pi-replete condition. As this result was not observed in walnut rootstocks, our data support the idea that at low soil fertility, walnut plant growth is limited more by nutrient uptake than by carbon supply ( Poorter and de Jong, 1999 ; Korner, 2003 ). Under these conditions, it is more advantageous for walnut saplings than for maize plant to allocate carbon assimilates to feed the AM symbiont ( van der Heijden and Horton, 2009 )."
} | 5,846 |
29473009 | PMC5816963 | pmc | 6,238 | {
"abstract": "Microbial diversity on earth is extraordinary, and soils alone harbor thousands of species per gram of soil. Understanding how this diversity is sorted and selected into habitat niches is a major focus of ecology and biotechnology, but remains only vaguely understood. A systems-biology approach was used to mine information from databases to show how it can be used to answer questions related to the core microbiome of habitat-microbe relationships. By making use of the burgeoning growth of information from databases, our tool “COREMIC” meets a great need in the search for understanding niche partitioning and habitat-function relationships. The work is unique, furthermore, because it provides a user-friendly statistically robust web-tool ( http://coremic2.appspot.com or http://core-mic.com ), developed using Google App Engine, to help in the process of database mining to identify the “core microbiome” associated with a given habitat. A case study is presented using data from 31 switchgrass rhizosphere community habitats across a diverse set of soil and sampling environments. The methodology utilizes an outgroup of 28 non-switchgrass (other grasses and forbs) to identify a core switchgrass microbiome. Even across a diverse set of soils (five environments), and conservative statistical criteria (presence in more than 90% samples and FDR q -val <0.05% for Fisher’s exact test) a core set of bacteria associated with switchgrass was observed. These included, among others, closely related taxa from Lysobacter spp., Mesorhizobium spp , and Chitinophagaceae . These bacteria have been shown to have functions related to the production of bacterial and fungal antibiotics and plant growth promotion. COREMIC can be used as a hypothesis generating or confirmatory tool that shows great potential for identifying taxa that may be important to the functioning of a habitat (e.g. host plant). The case study, in conclusion, shows that COREMIC can identify key habitat-specific microbes across diverse samples, using currently available databases and a unique freely available software.",
"conclusion": "Conclusions The COREMIC tool, by helping to mine multiple datasets fills an existing gap in the search for the core microbiome associated with a host or habitat. It allows for the development of a working hypothesis in the search for microbes well suited for a habitat or host-microbe interaction. It can also be used to confirm laboratory studies that have identified target microbes that might be important symbionts or thought to be associated with a specific habitat. In the case of plants, but not limited to them, the COREMIC approach can identify microbial targets that might be useful for plant growth promotion. An example of this would be the identification of diazotrophic bacteria that aid the growth of bioenergy grasses and help to serve the development of sustainable agricultural systems. This combined with the ongoing efforts of plant breeding and genetic modification would help to catalyze microbe-driven crop yield improvement while practicing environmental stewardship through reduced fertilizer use. Here we show the applicability of COREMIC in rhizosphere-associated microbes, but the overall concepts are translational across disciplines with interests in host-microbe and microbe-habitat relationships. The applicability of COREMIC for the identification of core genes and microbes has excellent potential to help understand the roles of microorganisms in complex and diverse microbial communities.",
"introduction": "Introduction Microbial diversity on earth is extraordinary, and soils alone harbor thousands of species per gram ( Hughes et al., 2001 ). Understanding how this diversity is sorted and selected into habitat niches is a major focus of ecology and biotechnology, but remains only vaguely understood. The advent of next-generation sequencing technologies now allow for the potential to make great leaps in the study of microbe-habitat relationships of highly diverse microbial communities and environments. The identity and functions of this overwhelming multitude of microbes are in the beginning stages of being described, and are already providing insights into microbial impacts on plant and animal health ( Berg, 2009 ; Evans & Schwarz, 2011 ; Clemente et al., 2012 ). Making use of the overwhelming amount of information on microbial taxa and habitats has enormous potential for use to further understand microbial-habitat relationships. Thus, the advent of new methods and approaches to utilize this data and describe microbiomes will benefit microbial ecology and biotechnology. Though variations exist, a core microbiome can be defined, conceptually, using Venn diagrams, where over-lapping circles and non-overlapping areas of circles represent shared and non-shared members of a habitat, respectively ( Shade & Handelsman, 2012 ). Typically, microbiomes identified in this manner are not statistically evaluated, or by nature, seek to answer specific hypothesis that are specific to an experiment. For example, studies often identify microbes associated with different plant growth stages, species, cultivars, and locations but rarely, if at all, mine databases or perform meta-analysis to statistically identify microbiomes across studies and experimental conditions ( Chaudhary et al., 2012 ; Liang et al., 2012 ; Mao et al., 2013 ; Mao et al., 2014 ; Hargreaves, Williams & Hofmockel, 2015 ; Rodrigues et al., 2015 ; Jesus et al., 2016 ; Rodrigues et al., 2017 ). Describing differences due to treatment or habitat conditions are informative in their own right, however, extending this framework to include an easy to use, and statistically robust tool to help in the mining of data from underutilized and burgeoning databases (e.g., the National Center for Biotechnology Information (NCBI), Ribosomal Database Project) can help transform the ecological study of microbes in their natural environment. Using the vast and growing databases of organism and habitat metadata will allow for both the testing and development of hypotheses associated with habitat-microbe relationships that were not formerly possible. To address the challenges described above, we developed COREMIC—a novel, easy to use, and freely available web tool to identify the “core microbiome”, of any well-defined habitat (e.g., plant root-zone) or niche ( Shade & Handelsman, 2012 ). This straightforward approach is a novel and powerful way to complement existing analysis (e.g., indicator species analysis (ISA) Dufrene & Legendre, 1997 ) by allowing for the use of data that is now overflowing among freely available databases. It seeks to determine the core set of microbes (core microbiome) that are explicitly associated with a host system or habitat. The ability to identify core microbiomes at this scale has great potential to describe host-microbe interactions and habitat preferences of microbes. A meta-analysis based case study was performed, combining diverse sequencing datasets derived from NCBI, to test for the occurrence of a core microbiome in the rhizosphere (root-zone) of switchgrass. Switchgrass is a US-native, perennial grass studied by many researchers, and thus has a growing database to mine for genetic information. Its widespread study is likely a result of its bioenergy potential, and the capacity of the grass to grow on marginal lands not dedicated to crops. Studies have identified different bacteria found in the root-zones of switchgrass ( Jesus et al., 2010 ; Mao, Yannarell & Mackie, 2011 ; Chaudhary et al., 2012 ; Liang et al., 2012 ; Mao et al., 2013 ; Bahulikar et al., 2014 ; Mao et al., 2014 ; Werling et al., 2014 ; Hargreaves, Williams & Hofmockel, 2015 ; Jesus et al., 2016 ; Rodrigues et al., 2017 ); however, there has been no integrative study of different datasets identifying the core microbiome in switchgrass rhizospheres. It was thus proposed to identify host-habitat relationships as a proof of concept for a core microbiome. In this paper we utilize a plant host to define a habitat, but theoretically any habitat and associated organisms could make use of COREMIC and its approach to identify a core microbiome.",
"discussion": "Discussion The case study showed how COREMIC can identify key habitat-specific microbes across diverse samples, using currently available databases and a unique freely available software. The core set of bacteria associated with switchgrass included, among others, closely related taxa from Lysobacter spp., Mesorhizobium spp , and Chitinophagaceae . The functional relevance of these bacteria related to switchgrass is currently unknown, but it is notable that these bacteria have been shown to produce bacterial and fungal antibiotics and promotethe growth of plants ( Kaneko et al., 2000 ; Kilic-Ekici & Yuen, 2004 ; Weir et al., 2004 ; Islam et al., 2005 ; Jochum, Osborne & Yuen, 2006 ; Ji et al., 2008 ; Park et al., 2008 ; Nandasena et al., 2009 ; Yin, 2010 ; Bailey et al., 2013 ; Degefu et al., 2013 ; Guerrouj et al., 2013 ; Madhaiyan et al., 2015 ). The analyses from the highly diverse data sets thus provided information that helps to greatly narrow down possibilities and thus set the stage for testing, using controlled studies, how the core microbiota potentially support or antagonize the function of a native grass. This novel toolkit is simple to use and supports use by a broad range of biological scientists, and is particularly relevant to those with expertise in their field but with limited bioinformatics background. Overall, in a dataset derived from a complex and diverse set of habitats and ecosystems, this tool was shown to pinpoint microbiota of the microbiome that might have important functional implications within their habitat or host. Methodological considerations in the use of COREMIC COREMIC performs a complementary analysis different from that of existing methods by using presence/absence data. For two groups (A and B) it checks whether (pre-determined percentage of) samples from group A have a non-zero value for the OTU. This allows scientists to operate without making assumptions about the PCR-based OTU relative abundances. This is considered a potential advantage of the method because it is unknown whether relative abundance of sequence data is representative of true relative differences between communities. It is well accepted that sequencing depth can affect the occurrence of rare OTUs in both relative and presence/absence data. Relative abundance analysis, however, would bias against rare OTUs, whereas presence absence equally treats abundant and rare OTUs since any count >1 (or as per the user chosen threshold) is treated as present. Using OTUs that are present in at least 90% of the interest group samples along with significance testing to define a core microbiome accounts for possibility that rare OTUs might be missed as being called present in some samples. Therefore, our proposed approach is relatively robust to sequencing depths. Further research, in this regard, will be aimed towards investigating other measures of OTU “presence”, namely the extent of exclusivity, consistency, or abundance of the group that is eventually determined to be a core microbiome. Sampling plots used in this study were located across a range of diverse environments to help create a backdrop of heterogeneity. While this diversity of habitat conditions ignores the potential for microbe-environment interactions that might be important for the plant-microbial relationship, it has the advantage of being a conservative approach with high veracity for defining a core microbiome regardless of habitat heterogeneity. The locations from which samples were grown (Michigan, Wisconsin, Virginia) were treated as independent to help isolate the overall habitat effect of switchgrass ( Werling et al., 2014 ; Jesus et al., 2016 ). When the effects of habitat are thought to be habitat specific, researchers can take this into account during the design and analysis using COREMIC. It is notable that the representation of an outgroup (multiple non-switchgrass species) is an important criteria and choice made by researchers, and is an approach that has both advantages and caveats. By definition, a habitat is defined by its differences from that of other habitats, and therefore the use of the outgroup is an important choice. A counter-argument for the current dataset might argue for exclusion of breeding lines of a cultivated grass (maize) as being unrepresentative of the grass outgroup. In our case, it was thought, a priori , that a diverse set of grasses would provide the best comparison; and no compelling argument was found that supported the exclusion of maize from the analysis. An implicit assumption was also made that the taxonomy of plant species (root-zone habitats) play an important role in determining root-zone microbial communities, an approach supported by extensive findings that different grass species associate with different microbial communities ( Kuske et al., 2002 ; Kennedy et al., 2004 ; Berendsen, Pieterse & Bakker, 2012 ; Chaudhary et al., 2012 ; Turner et al., 2013 ). So although there is a need for careful consideration of the experimental questions of interest when using COREMIC, this is a common, if not ubiquitous foundation of all experimentation and hypothesis testing. The results provide a statistically valid approach using freely available software to describe and define a core microbiome of switchgrass. The choice of the outgroup, furthermore, for determining a core microbiome is amenable to choice using deductive reasoning but ultimately limited by available data. This issue almost certainly limits inclusion of many functionally important rhizosphere microbes that could affect the growth of switchgrass. In this study, the proof of concept utilized a conservative approach to highlight the methodology across a diversity of geographies, soil types, and plant ages. The COREMIC tool as well as the multiple methods for defining a core microbiome (e.g., QIIME Caporaso et al., 2010 ), ISA ( Dufrene & Legendre, 1997 ) will always be defined by the expertise, and the nature of the hypotheses defined and defended by individual researchers. Core microbes The individual datasets described in this study had previously focused on identifying abundant microbes and differences due to experimental conditions. The current meta-analysis goes a step further to find common microbiota that are associated with switchgrass across the diverse experimental conditions. In fact, while Lysobacter would be identified as having significantly differential abundance as per the Man Whitney test, it would not be in the top 50 candidates (ranked as per FDR in Table S3 ) and likely missed from future testing. Clearly, our approach allows us to identify such candidates. The members of the Lysobacter genus, an identified core microbe of switchgrass, are known to live in soil and have been shown to be ecologically important due to their ability to produce exo-enzymes and antibiotics ( Reichenbach, 2006 ). Their antimicrobial activities against bacteria, fungi, unicellular algae, and nematodes have been described ( Islam et al., 2005 ; Jochum, Osborne & Yuen, 2006 ; Park et al., 2008 ; Yin, 2010 ). Strains of this genus, for example, have been used for control of diseases caused by bacteria in rice ( Ji et al., 2008 ) and tall fescue ( Kilic-Ekici & Yuen, 2004 ). Reports of their function thus support the idea that they may play an important role in switchgrass growth and survival. The core microbiome results thus support further research into the role played by this bacterium in the switchgrass rhizosphere. Similarly, members of the Mesorhizobium genus are well-known diazotrophs ( Kaneko et al., 2000 ) and previously shown to be symbiotically associated with switchgrass ( DeAngelis et al., 2010 ; Bahulikar et al., 2014 ) and legumes ( Weir et al., 2004 ; Nandasena et al., 2009 ; Degefu et al., 2013 ; Guerrouj et al., 2013 ). Another identified core microbiome taxa, soil-dwelling members of the Chitinophagaceae family are known to have β -glucosidase ( Bailey et al., 2013 ) and Aminocyclopropane-1-carboxylate (ACC) deaminase activities and ability to produce indole-3-acetic acid (IAA) ( Madhaiyan et al., 2015 ). These molecules and enzymes are well known for their effects on plant growth ( Zhao, 2010 ; Van de Poel & Van Der Straeten, 2014 ). The capacity to degrade cellulose might provide additional and readily available options to aid survival of these bacteria near switchgrass root zones during times of environmental stress. ACC deaminase and IAA production, in contrast, are potent plant growth modulators ( Glick, 2014 ) that could play a role in plant productivity and survival, especially under conditions of plant physiological stress. Though these examples above would need further study, they provide consistent examples describing how a core microorganism could play a role in determining plant function and growth. The power of the approach stems from the ability to identify the core microbes associated with a plant (or other habitat), and that can, with veracity, narrow down potentially important core microbes from otherwise hyperdiverse samples. From a technological standpoint, it is important to put the current approach into context with research before the metagenomics era. The search and identification of antagonistic plant growth promoting microbes has previously been tedious and labor intensive. Screenings of hundreds of microbes were used to cultivate and identify candidate microbes that might support (or deter) plant growth. In the case of beneficial microbes, even when identified under greenhouse conditions, the beneficial effects rarely translated into plant supportive growth under field growth conditions ( Babalola, 2010 ; Hayat et al., 2010 ). With the aid of hindsight and new knowledge suggesting the importance of the soil habitat and root-soil interactions in the development of growth promoting plant-microbial relationships, the approach used in this study reverses the focus (from top-down to bottom-up) to search for microbes that appear to already be naturally well-adapted to the root-soil habitats of interest ( Trabelsi & Mhamdi, 2013 ; Souza, Ambrosini & Passaglia, 2015 ). This process streamlines the search for suitable microbes from a daunting pool of thousands of bacterial taxa. Bacteria and fungi with well-known partnerships with members of the core microbiome, it would be expected, to be more readily adaptable to their native environment. Indeed, the concept of adaptability to an environment has been shown to be true for many types of microbes across the environmental spectrum, and has given rise to the concept of the niche ( Lennon et al., 2012 ). Existing tools (e.g., Corbata, MetaCoMET and QIIME) do not provide statistical significance for the taxa to be a core microbiome compared to the background using presence/absence data. The point of our method is to provide an approach that is definitive, rather than simply stating a set of microbe present in samples. Due to the lack of consensus in the scientific community about what a gold standard protocol/technology is for such studies (mainly due to the relatively early stages of the metagenomics field and technology still in developmental stages), one is limited (in one way or other) to sufficiently utilize the existing data. While our method of combining datasets has its limitation (like the granularity/species level information limitation from 16S, etc.), it still offers a powerful way to (i) understand the taxonomic distribution within samples (ii) mine multiple existing and often-diverse datasets (iii) generate hypothesis for future detailed experiments, and therefore, certainly a preferred alternative than not using existing data. The COREMIC tool provides an alternative and logical approach to help mine available datasets, in the search for core microbiomes associated with habitats that are ecologically and agriculturally important. Finally, each statistical test relies on different assumptions and has different strengths. COREMIC assumes that presence of a microbe, however high or low, can provide meaningful insight into potential host-microbe relationships. Therefore, it provides equal weightage for high and low abundant microbes within a sample. Since microbial abundance can be an important factor in a biological system, we recommend using COREMIC (presence/absence) in complement with other abundance-based methods (e.g., Mann Whitney test, ISA, etc.). Furthermore, while using diverse dataset has its strengths we suggest avoiding datasets where each dataset contains only a single (mutually exclusive) group. The user needs to consider similarities/differences between the datasets and the biological system while choosing an appropriate outgroup for their group of interest. It is up to the user to decide which thresholds make the most sense for their questions and hypothesis, but obviously a stricter threshold will have higher statistical-inferential veracity. Perhaps a combinatorial approach of selecting the top candidates from the different (presence/absence and abundance based) methods, picking microbes that show significant associations by multiple methods, and using the user’s biological expertise might better allow choosing candidates for future testing."
} | 5,374 |
37968339 | PMC10651889 | pmc | 6,240 | {
"abstract": "Identifying interspecies interactions in mixed-species biofilms is a key challenge in microbial ecology and is of paramount importance given that interactions govern community functionality and stability. We previously reported a bacterial four-species biofilm model comprising Stenotrophomonas rhizophila , Bacillus licheniformis , Microbacterium lacticum , and Calidifontibacter indicus that were isolated from the surface of a dairy pasteuriser after cleaning and disinfection. These bacteria produced 3.13-fold more biofilm mass compared to the sum of biofilm masses in monoculture. The present study confirms that the observed community synergy results from dynamic social interactions, encompassing commensalism, exploitation, and amensalism. M. lacticum appears to be the keystone species as it increased the growth of all other species that led to the synergy in biofilm mass. Interactions among the other three species (in the absence of M. lacticum ) also contributed towards the synergy in biofilm mass. Biofilm inducing effects of bacterial cell-free-supernatants were observed for some combinations, revealing the nature of the observed synergy, and addition of additional species to dual-species combinations confirmed the presence of higher-order interactions within the biofilm community. Our findings provide understanding of bacterial interactions in biofilms which can be used as an interaction–mediated approach for cultivating, engineering, and designing synthetic bacterial communities.",
"conclusion": "Conclusion In our study, the presence of the keystone species in viable form in close association with other cells was mandatory for the observed synergy. Community-level dynamics did not only arise from pair-wise interactions, but also from the influence of other species on many interacting pairs. Thus higher-order ecological effects beyond pairwise interactions may be key to understand interspecific interactions in simple microbial model communities. Establishing a deeper understanding of bacterial interaction will allow us to better predict the behaviour of bacteria, and to control and manipulate bacterial biofilms for environmental, industrial and clinical purposes. Fluctuating environmental conditions including nutritional status alter the dependency between the two bacterial strains and thus the results obtained in this study should be interpreted with caution.",
"introduction": "Introduction Biofilms are increasingly recognised as an important concern for multiple industries, affecting food production and safety, water supply, health, industrial processes, and the marine sector where the presence of biofilms causes economic impact of billions of USD per year [ 1 ]. Almost all biofilms in natural and industrial settings are composed of multiple and often diverse species that interact with each other and with the environment in a variety of ways as part of the “struggle for existence” [ 2 , 3 ] and establish several associations ranging from positive interactions (e.g., cooperation +/+ and commensalism +/0 to competition −/−, amensalism 0/−, and exploitation +/− [ 4 – 6 ]. Multispecies biofilms in the dairy industry can contain both pathogens and food spoilers. These biofilms have implications for the safety and quality of food products and economy overall because of their association with enhanced production of spoilage enzymes and toxins [ 7 – 9 ]. Bacteria recovered from food contact surfaces after cleaning and disinfection (C&D) have indeed been shown to interact with each other in a variety of ways that may have implications for persistence and tolerance of these biofilms against disinfectants [ 10 – 12 ]. Despite the awareness of the significance of bacterial interactions in biofilms and their consequences, our current understanding of interspecies interactions – or even the principles governing these interactions in general – is limited, which is mainly because bacterial interactions are complex to study [ 13 ]. Outcomes of bacterial pairwise interactions have been used to predict the structure and function of a number of simplified bacterial communities [ 14 , 15 ]. However, pair-wise interactions do not take into account another type of interaction, termed higher-order interaction, in which the interaction between two species is modulated by one or more other species [ 16 ]. Community dynamics are often affected by one or few individual species termed keystone species. A keystone species is a species which, regardless of its frequency, has a significant effect on the ecology, survival and function of other species [ 17 ]. Thus, studying variability and strength of both pair-wise and higher-order interactions is imperative to understand drivers of species coexistence in diverse communities [ 18 ]. This is not only fundamentally interesting, but this knowledge is important to predict stability and functionality of the community, its evolutionary dynamics and bottom-up biological functions in a range of contexts including controlling biofilms on food contact surfaces. In our previous work [ 19 ], we characterised 140 reproducible four-species biofilm communities on stainless steel (SS) that comprised bacteria previously recovered from the surface of a dairy pasteuriser after routine industrial C&D practices [ 20 ]. Out of the 11 four-species combinations that showed synergy (higher biofilm mass in co-culture than the sum of the monoculture biofilm masses) in four-species biofilms, five combinations had three species in common: Stenotrophomonas rhizophila , Bacillus licheniformis , and Microbacterium lacticum . A study on bacterial ecology of biofilms on the surface of a milking machine also reported coexistence of these three species in multispecies biofilms [ 21 ]. Strong synergy in biofilm formation was observed in a four-species biofilm community that included these three species and Calidifontibacter indicus [ 19 ]. However, the re-organisation of bacterial interactions between pairwise cultures and larger communities remained largely unknown. From an ecological perspective, understanding the role of individual species in observed synergy, stability, and community assembly is crucial for a mechanistic understanding of microbial synergistic interactions. Here, we used a bottom-up approach and disentangled the interspecies interactions and growth dynamics in the four-species community by analysing all pairwise and higher order interactions (e.g., three-species interactions and four-species together) between the component species. Overall, this is the first study on dairy isolates which provides an in-depth understanding of the role of various social interactions – from commensalism to exploitation – and assesses the significance of keystone species within a multispecies biofilm community for stability, co-existence and perhaps better survival. Knowledge on these specific bacterial interspecific interactions and the role of keystone species may translate to other bacterial interactions and deepen our understanding of bacterial ecology in general.",
"discussion": "Discussion In mixed biofilm communities, bacterial species often gain fitness advantages, evident through enhanced growth and increased biofilm mass when co-existing with other species. Several factors contribute to this advantage. One of the factors is the facilitated exchange of nutrients and waste products within these mixed communities, which can provide distinct growth benefits to specific species [ 27 ]. Additionally, interactions between species can modulate the biofilm’s architecture, thereby improving nutrient availability [ 28 ]. Our findings further substantiate the presence of dynamic social interactions among species within a single biofilm community. These interactions can have varied implications for the growth and matrix production of the resident species. SS coupons were used in our trial to simulate the dairy pasteurizer environment from which the species were originally isolated. The four-species combination was grown in both BHI and SM. The growth dynamics in SM are more relevant for the dairy industry, and using different media highlights the consistency of our findings. SM was not used in all trials (e.g., pairwise combinations and trios) due to milk protein coagulation preventing biofilm mass measurement, allowing only for cell counts. Hence, SM and BHI were used exclusively for cell population dynamics in the four-species combination, while BHI was used to determine cell counts and biofilm mass for other combinations. The results from all possible pairwise interactions among the four species reaffirmed the concept of keystone species in communities. A disproportionately large effect of M. lacticum on growth of all three species and on overall synergy in biofilm mass formed by the four-species community strongly indicated the role of M. lacticum as a keystone species. Keystone species in a biofilm community–regardless of their proportion – often serve as a trigger of biofilm formation in other species as well as a metabolic facilitator or protector [ 29 – 31 ]. The significance of keystone species in promoting multispecies biofilms [ 31 ] and conferring anti-microbial tolerance to the community members [ 32 ] has been reported. Exclusion of a single strain – Actinobacteria ( Rhodococcus or Microbacterium ) - from a 62-strain community was shown to significantly affect the community diversity and structure [ 33 ]. Some keystone species (e.g., Enterococcus faecalis, Porphyromonas loveana and Dialister pneumosintes ) are important drivers of bacterial community composition and their absence affects the abundance of several other bacterial species due to their role in stimulating the growth of other bacteria [ 34 , 35 ]. In our combination, it is a possibility that M. lacticum is the only species which efficiently adheres and forms biofilm on SS and other species interact with M. lacticum by adhering to its surface and B. licheniformis plays an important role in building up the matrix. When M. lacticum was replaced with its CFS in the SR-ML and BL-ML combinations, there was no observed change in the biofilm mass of S. rhizophila and B. licheniformis . In a study examining biofilms formed by bacteria isolated from soil, no noticeable effect of the CFS from one bacterium on another was observed in various combinations [ 23 ]. One explanation is that the CFS of M. lacticum used in this study was derived from overnight growth of monoculture M. lacticum , whereas in our model biofilm, M. lacticum impacted the growth of S. rhizophila in co-culture conditions in a structured biofilm environment. Certain metabolites that mediate interaction can moreover be unstable and degraded in CFS before administration and thus the dynamics of exposure of partner species to metabolites of the other species, produced in monoculture vs co-culture, could be different. A significant increase in the biofilm mass of B. licheniformis in the presence of S. rhizophila without any significant change in its cell count may be related to stress response. EPS is often produced as a stress-response strategy in bacterial biofilms, and it is exploited by non-EPS producing strains to get protection or to fulfil certain nutritional needs [ 36 , 37 ]. Here, we also provide evidence of higher-order interactions in the biofilm community. CFS of S. rhizophila and B. licheniformis possibly contained metabolites which induced cell growth or biofilm formation of either species in the presence of M. lacticum . Another example of higher-order interaction was the effect of C. indicus on two dual-species combinations: SR-BL and BL-ML. Keeping in mind a pronounced interaction leading to a greater increase in biofilm mass in the dual-species combination SR-BL, it could be inferred that the effect of CFS from B. licheniformis and S. rhizophila on SR-ML and BL-ML, respectively, was related to the interaction between the two species ( S. rhizophila and B. licheniformis ) which was further enhanced in the presence of M. lacticum . Another possibility is the generation of new niches by bacteria through secretion of molecules that possibly alter the pH of the microenvironment and thereby affect the growth of both themselves and also other microbes [ 38 ]. According to ‘coexistence theory’ which is based on pH sensitivity of bacterial communities, when a certain type of bacteria dominate, the pH is biased to the optimum value of the dominant bacteria, which strongly suppresses the growth of other type of bacteria [ 39 ]. In our trial, CFS fractions of B. licheniformis or S. rhizophila (pH > 8) might have altered the pH, creating an alkaline environment, which might have favoured the growth of either of these species suppressing M. lacticum . Bacterial community interaction networks are central for understanding the structure and function of microbial communities in natural and industrial settings. It has been a matter of debate over the last one decade whether pairwise interactions are good determinants of community assembly [ 40 ] or higher-order interactions should be taken into account to explain community structure [ 41 , 42 ]. We observed that the outcomes of pair-wise interactions did not provide insights into specific interactions in which a pair-wise interaction was mediated by a third species (e.g., SR-BL-CI). We confirmed the presence of interspecies interaction network among the four species which probably equilibrated all negative pair-wise interactions into a ‘competitive balance’. Nevertheless, there is no evidence that strong higher-order positive effects emerge when specific species pairs, interacting negatively, also interact with other species, which is in line with a previous finding [ 43 ]. A study on Zebrafish gut bacterial communities [ 44 ] and on bacteria isolated from Caenorhabditis elegans intestines [ 45 ] showed that bacterial competitive interactions in pairs could not be used to predict species abundances in more complex communities because higher-order interactions dampen pairwise competition. Overall synergy of the four-species community was mainly a result of the growth-promoting effect of M. lacticum on other species which further mediated pair-wise interactions. C. indicus stimulated growth of B. licheniformis which mediated interaction between S. rhizophila and M. lacticum . The interaction network between the four-species based on our findings is shown in Fig. 7 . Fig. 7 A schematic presentation of bacterial interspecies interactions in a four-species biofilm community on stainless steel in brain-heart-infusion medium comprising Stenotrophomonas rhizophila (SR), Bacillus licheniformis (BL), Microbacterium lacticum (ML), and Calidifontibacter indicus (CI). M. lacticum is shown to induce growth in all other species, whereas its own growth is negatively affected by B. licheniformis and C. indicus . S. rhizophila is shown to have a neutral interaction with B. licheniformis . C. indicus is shown to be exploited by B. licheniformis as it induces growth of B. licheniformis with a negative effect on its own growth. S. rhizophila , B. licheniformis , and C. indicus can be seen to mediate interactions in BL-ML, SR-ML and SR-BL, respectively. Bacterial ratios in SM- and BHI-based four-species biofilm communities did not vary much except the behaviour of M. lacticum , which achieved higher cell numbers and remained stable in SM for a longer period of time. Milk-adapted strains of M. lacticum have been reported to grow to higher cellular densities in milk compared to other media [ 46 ]. Bacterial interspecies interactions depend on the environment and the availability of nutrients. The relative abundance of a bacterial strain at the beginning of co-cultured incubation is not predictive of its colonisation success at later stages [ 33 ]. M. lacticum was exploited in the community and other species strongly depended on it and gained growth advantages. It is important to highlight that in the mixed-species biofilm on SS in BHI, M. lacticum was the predominant species until 12 h. However, after this point, its growth significantly decreased while that of S. rhizophila and B. licheniformis notably increased. This suggests that the advantages the latter species experienced might have been influenced by M. lacticum ’s growth dynamics. Later on other species outcompeted M. lacticum and dominated the community. Looking at the pH value of the planktonic fractions of the four-species community (pH = 8) and M. lacticum monoculture (pH = 6), it is possible that high pH of the medium caused by the growth of B. licheniformis and S. rhizophila affected the growth of M. lacticum . Further studies at the transcriptional level are required to understand the gene expression patterns of each species in single and different combinations to gain a better understanding of metabolic interactions or any other associations among these community members. However, it appears that targeting M. lacticum will disintegrate the observed synergy and this is the point where strategies targeting the whole communities can be developed once the mechanistic basis of these interspecies interactions are understood."
} | 4,331 |
36506194 | PMC9730311 | pmc | 6,241 | {
"abstract": "The design and utilization of polymers with healing capability\nhave drawn increasing attention owing to their enhanced chain mobility\nand opportunity to heal minor cracks in composites. Rehealable thermoset\npolymers promise reduction in the maintenance cost and thus prolonged\nlifetime, reshaping, and recyclability. Introducing reversible covalent\nbonds is the mainstay strategy to achieve such plasticity in crosslinked\npolymers. Herein, we report a dynamic epoxy, which includes associative\ncovalent adaptive networks (CANs) based on disulfide exchange bonds.\nEpoxy resin is chosen to study rehealing, as it is one of the most\ncritical thermosetting polymers for various industries from aerospace\nto soft robotics. This study enlightens us about not only the consequences\nof CANs in the epoxy but also various factors such as soft segments\nand carbon nanotubes (CNTs). Epoxy dynamic networks are investigated\nin an attempt to explore the synergistic effect of the soft-segmented\nresins and CNTs on the healing and reshaping characteristics of epoxy\nnetworks along with varying stiffness. This research discusses epoxy\ndynamic networks in three main aspects: crosslink density, CAN density,\nand CNTs. Introducing soft segments into the epoxy network enhances\nthe healing efficiency due to the increased chain mobility. A higher\nCAN density accelerates network rearrangement, improving the healing\nefficiency. It should also be noted that even with a low weight fraction\nof nanotubes, CNT-reinforced samples restored their initial strength\nmore than neat samples after healing. The tensile strength of dynamic\nnetworks is at least 50 MPa, which is significant for their utility\nin primary or secondary structural components.",
"conclusion": "4 Conclusions In this study, healing\nand reshaping mechanisms of epoxy dynamic\nnetworks with reversible covalent adaptive networks are examined based\non CAN density and crosslink density together with CNT inclusion.\nVarious combinations of neat and CNT-reinforced epoxy dynamic networks\nare analyzed carefully to identify their thermomechanical and healing\ncharacteristics. It is crucial to point out that the tensile strengths\nof epoxy dynamic networks are at least 50 MPa even with a soft segment.\nHigher flexibility, lower crosslink density, and higher CAN density\nare found to be favorable for an enhanced healing behavior. VT samples\nwith only a hard-segmented resin (DGEBA) exhibit a healing performance\nthat is less than ideal. Introducing soft segments (DGEPG) into the\nexisting network enhances the healing efficiency remarkably. Furthermore,\nit is proven that CNTs implement a lower T g and higher mobility and thereby a higher healing efficiency. In\naddition to healing, epoxy dynamic networks display a repetitive reshaping\ncapability, promoting the recyclability for environmental protection.\nThis study offers a better understanding of tuning the stiffness of\nepoxy along with healing and reshaping, promising a reduced maintenance\ncost and prolonged lifetime for the industrial applications of epoxy.",
"introduction": "1 Introduction The discovery of self-healing\nmaterials has driven researchers\nto investigate reversible bonds in polymeric networks especially in\nthermoset polymers, which enable new possibilities for various potential\napplications. Among thermoset polymers, epoxy is a widely used resin\ndue to its high mechanical performance, durability, and creep resistance.\nApart from their outstanding mechanical performance, epoxies also\nexhibit good thermal properties and high chemical resistance. However,\nthere are still challenges to overcome in thermoset epoxy resins,\nsuch as their lack of recyclability and reprocessability. 1 − 3 Epoxies are subjected to considerable stresses, high temperature,\nand pressure, which may harm the structural integrity of the composite\nand reduce the operation lifetime. In the case of an even minimal\ndamage, it is not possible to repair thermosets due to their irreversible\ncovalent bonds. Such a scenario is far from being industry-friendly\nsince the damaged composite part is replaced each time with an excessive\nmaintenance cost. 2 Using microcapsules\ninside the polymer has been researched to reduce this cost and unexpected\nrisks. In this concept, the microcapsules get triggered with crack\ninitiation/propagation and release the uncured resin inside the capsule\nto heal the crack. 4 , 5 The drawback of microcapsules\nis that the process is not reversible and self-healing can be performed\nonly one time. In addition, it is challenging to maintain microcapsules\nstabilized during the whole operation time of the composite. Polymers\nwith reversible crosslinks have been proposed broadly in the past\ntwo decades, including covalent and noncovalent reversible bonds. 6 − 11 The problem with noncovalent interactions is that they are prone\nto be fragile, which cannot survive under large strains. 6 Furthermore, there are systems of dissociative\nreversible covalent adaptive networks (CANs) that depolymerize above\na critical temperature and reform new covalent bonds each time. 6 However, a dissociative CAN system, Diels–Alder\nas a typical example, is not desirable since it is difficult to maintain\nthe network integrity under such a sudden viscosity drop during the\nhealing process. 12 , 13 Associative covalent adaptive\nnetworks overcome the challenges\nsuch as reusability and depolymerization owing to their constant crosslink\ndensity retaining the integrity of the whole structure during the\nhealing process. Among different CAN systems such as transesterification, 7 , 9 transcarbamoylation, 14 imine exchange\nreaction, 15 , 16 etc., disulfide metathesis stands out as\na leading mechanism within its capability of bond exchange reactions\nat relatively lower temperatures. 1 , 2 , 7 , 8 , 17 − 20 Li et al. 19 suggested that embedding\ndisulfide dynamic links in epoxy resin, including soft segments, can\nrecover its original strength at least 65% at 80 °C. Despite\nthe achieved high healing efficiency, it should be noted that such\na concept demonstrates high flexibility and low mechanical strength,\nrestricting their utility in advanced composites. Canadell et al. 21 developed an epoxy with disulfide links, which\nis capable of healing around 80%. The original strength of the sample\nwas limited to 0.6 MPa at most, which cannot be used for any load-bearing\nstructural component. The improvement in the healing of epoxy-based\nsamples was attributed to the adequate mobility for the macroscopic\nflow using DER732 epoxy resin with low viscosity. Similarly, Lafont\net.al. 22 proved that the increased chain\nrigidity hampers the healing kinetics, reducing the probability of\ndisulfides to come into contact. On the other hand, Luzuriaga et.\nal. 2 synthesized a recyclable epoxy resin\nwith a tensile strength of 90 MPa recovering its strength nearly fully\nafter reshaping. In the case of self-healing, this study only monitors\na small scratch on the surface of the epoxy instead of a complete\nfailure. Healing of an epoxy sample broken completely in half with\na high tensile strength is still open to discussion. To the best of\nour knowledge, healable epoxy networks studied in the literature either\nshow low strength and elastomer-like behavior 7 , 21 or\nquite the opposite, a very brittle nature. 23 , 24 There is a need to understand the comprehensive properties with\nidentical crosslink density and CAN density. Therefore, tailoring\nthe composition of epoxy networks enables a wide range of products,\nfrom rubbery to rigid components that are rehealable and recyclable\nand can be utilized to meet industrial demands. 19 While producing rehealable epoxy networks with adjustable\nstiffness,\nit is essential to preserve the mechanical strength in the meantime.\nCarbon nanotubes (CNTs) come up as a promising solution to increase\nthe mechanical strength 25 − 27 and even offer a healing mechanism\nwith multiple stimulation options. 1 , 14 Bonab et.al. 14 fabricated a CNT-reinforced polyurethane network\n(TPU) with adaptive covalent bonds and indicated that around 45% of\nthe original strength can be recovered when exposed to microwave radiation.\nIt is noteworthy that the study examines transesterification and carbamate\nexchange reactions in the TPU system and the tensile strength is limited\nup to 10 MPa. In addition, there have always been attempts to discover\nthe effects of CNTs on chain mobility and polymerization. 28 , 29 For instance, the glass transition temperature is found to be dependent\non the existing CNT dispersion media, surfactants, and polymer chemistry. 28 Simulations 20 , 30 and experiments 29 , 31 reveal that CNTs tend to decrease the glass transition temperature\nand trigger the activation of the bond exchange reaction at lower\ntemperatures. 1 , 14 Such a decrease in the glass\ntransition temperature is ascribed to the reduced crosslinking tendency\nin the epoxy network in the presence of CNTs. 31 Similarly, Miyagawa and Drzal 32 analyzed\nthe effect of CNTs on the thermophysical properties of the epoxy and\nobserved the same decrease in glass transition temperature linearly\nby increasing CNT weight ratio. On the other hand, CNTs display a\nconstructive influence on the mechanical strength since CNTs possess\na high Young’s modulus as a combination of individual moduli\nof each graphene sheet and the van der Waals forces. 25 , 26 To benefit from CNT strength, it is crucial to disperse CNTs in\nthe polymer homogeneously, prepare a stable suspension, and prevent\nagglomerations, which can act as stress concentration points under\nan applied load. 32 , 33 Applying physical functionalization\nwith a nonionic surfactant is one of the effective methods to improve\nthe interfacial interactions between the CNTs and polymer, enabling\nthe suspension to become more stable. 34 , 35 Consequently,\nthis study elaborates on the effects of more flexible\nsegments and CNTs on epoxy dynamic networks concerning the trade-off\nbetween comprehensive properties and healing efficiency. Associative\ncrosslink adaptive networks are embedded into the epoxy resin with\ndisulfide dynamic bonds. Rehealable epoxies are synthesized based\non different compositions of resin-to-disulfide molar ratios without\nany catalyst, as catalysts may cause instability and toxicity. 2 , 6 , 36 For the resin, two different\ntypes of commercial resins are used, including hard and soft components.\nInstead of using only a conventional hard segment resin, it is aimed\nto tune the stiffness of the epoxy dynamic networks using soft segments.\n\n2.2.2. Synthesis of the Soft Segment-Introduced Epoxy Dynamic\nNetworks DGEPG as the soft segment is mixed with DGEBA in\ndifferent molar ratios at 80 °C. The weighed AFD is then added\nto the solution, and the whole solution is mixed at 80 °C for\n40 min. Herein, to study the effect of soft segments on the healing\nprocess, the molar ratio of the epoxy resin to the disulfide hardener\nis fixed at 3:2. Then, degassing is performed at 80 °C for 2\nh to get rid of bubbles. The resulting epoxy (R-VT) is cured at 150\n°C for 10 h in an oven.",
"discussion": "3 Results and Discussion 3.1 Morphological and Thermomechanical Properties\nof Epoxy Dynamic Networks The two contradicting aspects crosslink\ndensity and CAN density inside the dynamic network should be optimized\nto enhance the healing performance while retaining the mechanical\nstrength. A relationship between the performed modifications and healing\nbehavior is established by the morphological and thermomechanical\ncharacterization of epoxy networks. 3.1.1 Fourier Transform Infrared Spectra FTIR is conducted with a Thermo-Scientific-Nicolet 6700 to ensure\nthat the samples are fully cured. Figure 2 c displays the disappearance of the C–H\nstretching of epoxide rings at 914 cm –1 , 37 showing that the curing process is highly completed.\nThe decreasing intensity in another characteristic vibration peak\nof the epoxide group at about the 845 cm –1 band 19 , 37 also proves that curing is complete. The peaks at 1510 and\n1580 cm –1 bands remain constant and correspond to\nthe aromatic rings. 19 , 38 The band at 1100 cm –1 is attributed to the vibration of the C–O–C segment\nfrom the aliphatic ether. 19 , 39 , 40 The broad absorption band centered at about 3400 cm –1 shown in Figure 2 d corresponds to the hydroxyl groups formed by ring opening reactions\nduring the curing process. 19 Also, the\npeak at 2900 cm –1 is related to the aliphatic C–H\nstretching vibration. 39 Full-scale FTIR\nspectra of Ref-Ep and VT samples can be seen in Figure S1 . 3.1.2 Thermogravimetric Analysis TGA\nis carried out using a Discovery TA Instruments device in a nitrogen\natmosphere to obtain the degradation temperature and hence the overall\nthermal stability of epoxy networks. A heating rate of 10 °C/min\nis applied from 25 to 400 °C. Compared to Ref-Ep samples, Figure S4 , TGA profiles of dynamic networks have\nthree significant degradation steps characterized by their derivatives, Figure S3b . The first weight loss, around 250\n°C, is assigned to the decomposition of oxygen-containing groups.\nAround 300 °C, disulfide bonds begin to decompose. After 350\n°C, a sharp degradation is observed due to epoxy resin pyrolysis. 19 Figure 3 a outputs a decrease in the thermal stability of epoxy\ndynamic networks as the CAN density increases. The presence of disulfide\nspecies results in slightly poor degradation behavior since they have\na lower bond energy than that of carbon–carbon bonds. 24 Less energy is required to decompose the chemical\nbonds when more S–S bonds are present in the epoxy network.\nVT-3 exhibits the lowest decomposition temperature owing to its higher\nCAN density, wherein VT-1 is the most stable network thermally. 19 Since VT-2 and VT-3 samples are synthesized\nwith a nonstoichiometric ratio, the effect of crosslink density is\nalso studied to confirm the major reason for the depression in degradation\ntemperature. Stoichiometric and nonstoichiometric reference epoxy\nnetworks, Ref-Ep-1 and Ref-Ep-2, respectively, are subjected to TGA.\nConsidering the result in Figure S4 , CAN\ndensity is determined to be the driving force of the change in the\ndegradation behavior of VT samples. Likewise, the soft segment-introduced\nepoxy dynamic network (R-VT-1) illustrates lower thermal stability\nthan that of its counterpart, VT-2, as given in Table 3 . The lower thermal stability is related\nwith the soft segments since DGEPG starts decomposing earlier than\nDGEBA due to its aliphatic polymer chain backbone having less heat\nresistance. 41 In the case of CNT-reinforced\nnetworks, thermal decomposition starts earlier than for their counterparts, Figure 3 b. This shows that\nreinforcing the dynamic networks with CNTs increases the heat diffusion\nand hence initiates a slightly faster degradation. 42 The lower decomposition temperature of the CNT-reinforced\nepoxy can also be linked to the reduced crosslink density, as presented\nin Table S1 . Such a loss in crosslink density\nis attributed to the decreased crosslinking tendency 31 due to the penetration of CNTs in free volumes and a large\namount of interphase region between the CNTs and polymer. 30 , 32 Figure 3 TGA\nweight loss thermographs of epoxy dynamic networks (a) with\nrespect to increasing CAN density and (b) CNT-reinforced samples and\ntheir counterparts. Table 3 Thermal Properties of Epoxy Networks sample VT-2 VT-3 CNT-VT R-VT-1 R-VT-2 CNT-RVT T g (°C) 122.4 109.8 111.3 46.3 35.9 44.4 1% weight loss (°C) 268.88 264.94 240.85 220.3 5% weight loss (°C) 301.72 289.09 284.46 282.21 Δ c p (J/g·K) 0.0069 0.0094 0.0105 0.0110 0.0127 0.0113 3.1.3 Differential Scanning Calorimetry DSC analysis is conducted using a Discovery TA Instruments device.\nMeasurements are recorded at a heating rate of 10 °C/min from\n−40 to 120 °C for R-VT samples and from 40 to 200 °C\nfor VT samples under a nitrogen atmosphere. DSC curves demonstrated\nin Figure 4 correspond\nto a second heating after cooling. The curing state of the epoxy samples\nis also addressed from the first heating ramp of nonisothermal DSC\nand from isothermal DSC. According to Figure S5f , no exothermic peak is observed, confirming that the process time\nand temperature are enough for the complete curing of epoxy samples. T g values are determined from the midpoint of\nthe step in the heat flow curves, Table 3 . The glass transition temperature for the\nRef-Ep-2 sample is detected as 120.5 °C from Figure S5e . Figure 4 DSC curves of (a) crosslinked and (b) soft segment-introduced\nepoxy\ndynamic networks. Figure 4 a clarifies\nthat the T g value of the epoxy dynamic\nnetwork decreases as the disulfide content increases. A higher amount\nof disulfide exchange bonds imparts easier segmental mobility since\nthey break and reform in numerous sites. According to Table 3 and Figure 4 b, it is clear that R-VT samples possess\na lower glass transition trend compared to that of VT samples. The\nsignificant decrease in T g values is associated\nwith increasing soft segment molar ratio since it refers to more aliphatic\nbranching chains and higher flexibility. 41 The lower glass transition temperature in CNT-reinforced samples\nshows the enhanced polymer segmental mobility. 28 , 31 The presence of CNTs in the free volume of the polymer reduces the\ncrosslink density, 31 leading to a decrease\nin T g . Another reason for such a decrease\nin the glass transition temperature of CNT-reinforced epoxy dynamic\nnetworks is the high thermal conductivity. It is assumed that introducing\nCNTs into the epoxy matrix might increase the internal heat diffusion\ninside the polymer by generating three-dimensional (3D) heat conduction\npathways. 42 , 43 Such effects of CNTs on the glass transition\ntemperature are well known from previous studies. 1 , 30 , 44 The mobility of the chains can also\nbe inferred from the heat capacity\nincrease (Δ c p ) at glass transition\nas a measure of the fraction of polymer chains involved in glass transition. 45 − 47 The calculation of Δ c p from DSC\ncan be found in the Supporting Information . Among VT samples, Table 3 , the heat capacity increase of VT-2 is found to be the lowest,\nas it forms the strongest crosslinked network, Table S1 , having the stoichiometric ratio. VT-3 shows a higher\nΔ c p , indicating that a higher amount\nof polymer chains is free of molecular motion and participates in\nglass transition. The higher Δ c p values in R-VT samples are interpreted as the lower restriction\nof segmental motion as a result of the increased aliphatic chains.\nCNT samples revealed a higher Δ c p than that of their counterparts, referring to the decreased crosslink\ndensity in both cases. Having a higher Δ c p offers a higher healing efficiency since mobility and healing\nare proportional. 3.1.4 Dynamic Mechanical Analysis DMA\nis performed in the tension mode using a Mettler Toledo DMA/SDTA model\nto address the viscoelastic behavior of epoxy dynamic networks. DMA\nis applied to 3 × 5 × 20 mm 3 (thickness, width,\nlength) samples with a force amplitude of 5 N, a displacement amplitude\nof 5 μm, and a frequency of 1 Hz. The loss modulus profile of\nall epoxies follows a lower trend compared to that of storage modulus,\nverifying the viscoelastic behavior. Furthermore, all samples follow\na stable storage modulus trend in both glassy and rubbery states. Figure S7 shows that CNT-reinforced samples\nexhibit a slightly higher storage modulus than that of their neat\ncounterparts. The higher initial storage modulus of the CNT-reinforced\nepoxy networks refers to the polymer chain motion restriction due\nto the particle–particle and particle–polymer interactions\nat room temperature. 48 Increasing the temperature\nfor CNT-reinforced samples is expected to contribute to the internal\nheating of the polymer faster and trigger the Brownian motion of CNTs,\npromoting mobility easier than for neat samples. 49 R-VT-1 displays a lower storage modulus than that of VT-2,\nindicating its less-rigid structure. 50 The\nflexibility in R-VT samples promises higher healing performance and\nimproved toughness. In Figure 5 b, comparing the peak values of tan δ (loss factor)\ncurves, VT-3 and CNT-VT samples have a higher loss factor than that\nof VT-2. R-VT-2 and CNT-RVT also exhibit a slightly higher loss factor\nthan that of the R-VT-1 sample. This indicates higher healing potential\nsince lower viscosity means more segmental motion and bond exchange. Figure 5 (a) DMA\ncurve of the VT-2 sample representing storage and loss\nmodulus and (b) tan δ curves. 3.1.5 Stress Relaxation Stress relaxation\nis conducted using a DMA Q800, TA Instruments. The samples with a\nsize of 3 × 13 × 35 mm 3 are measured at a strain\nof 1% in the linear region, and the relaxation of epoxy networks is\nmonitored as a function of time for 1 h. Time- and temperature-dependent\nbehaviors of epoxy networks are studied carefully to validate the\nrelaxation behavior of dynamic epoxy networks with reversible covalent\nadaptive networks. The studied temperature profiles are chosen to\ncompare the relaxation behavior of dynamic networks below and above\ntheir glass transition points. Figure 6 shows that epoxy dynamic networks are able\nto flow in all cases and attain a less stretched form due to the disulfide\nexchange mechanism. Comparing Ref-Ep and VT-2 samples, there is no\nobvious relaxation behavior in the Ref-Ep-2 sample, signifying the\nimportance of disulfide exchange bonds in the epoxy network. Both\nVT-2 and R-VT-1 samples begin to relax below their T g , even though it is not a complete relaxation. This is\nlinked to topology freezing temperature ( T v ), which is defined as the transition from solid to liquid as a result\nof bond exchange. 2 , 24 For dynamic thermoset polymers, T v is lower than T g , 51 confirming the relaxation behavior\nin VT-2 and R-VT-1 samples, as the bond exchange may begin below T g . Figure 6 b depicts that G / G 0 values reach zero for both VT-2 and R-VT-1 samples,\nimplying full stress relaxation above T g . This is related to the increased bond exchange rate and segmental\nmotion above T g . 2 For the samples showing complete relaxation, the relaxation times\nare defined as the time required to relax 63% of initial stress 2 or to obtain G / G 0 = 1/ e with reference to the Maxwell\nmodel for viscoelastic fluids. 19 , 52 The achieved relaxation\ntimes are found to be 4.05 min for VT-2 at 150 °C and 3.34 min\nfor R-VT-1 at 80 °C. The R-VT-1 sample displays a higher relaxation\nrate than that of other samples, which can be attributed to the flexible\nchain backbone due to the soft segment portions. Consequently, the\ntemperatures in healing and reshaping procedures are decided to be\nadequate for the network to relax completely and gain mobility. Figure 6 Normalized\nstress relaxation curves of the epoxy reference and\ndynamic networks (a) below T g and (b)\nabove T g . 3.1.6 Solvent Resistance The chemical\nresistance of epoxy networks is examined to ensure that the rehealable\nepoxy network does not dissolve in the presence of a solvent yet and\nshows stress relaxation still. In a similar manner to the previous\nstudies, 23 , 53 a part of VT-2 and R-VT-1 are immersed in\ntrichlorobenzene for 4 h first at RT and then at 80 °C with continuous\nstirring, Figure 7 .\nNo alteration is monitored in epoxy dynamic networks even after 24\nh similar to conventional epoxy composites. This indicates the excellent\nsolvent resistance and crosslinked network of dynamic epoxies with\ndisulfide exchange bonds. Figure 7 (a) VT-2 sample in trichlorobenzene after 24\nh at RT (b) with continuous\nstirring at 80 °C and (c) VT-2 sample in trichlorobenzene after\n4 h at 80 °C. 3.1.7 Micro-tensile Test This research\nexamines varying crosslink and CAN densities with the assistance of\nsoft segments and CNTs to achieve the required polymer mobility without\nsacrificing strength. Tensile tests are conducted using a Psylotech\nmicro-tensile testing machine and 10 kN load cell, until the complete\nfailure of the samples. The testing speed is set as 100 mm/min, and\nthe samples are prepared in a dumbbell shape of 4mm thickness and\n60mm length, as shown in Figure 8 c. Table 4 illustrates the average tensile strength\nfor at least three samples in each batch. VT-1 having the stoichiometric\nratio exhibits the highest mechanical strength as expected due to\nits higher crosslink density, wherein increasing the CAN density leads\nto a decrease in tensile strength for VT-2 and VT-3 proportionally. Figure 8 (a) Stress–strain\ncurves of crosslinked, soft segment-introduced,\nand CNT-reinforced samples; (b) sample cut in half (1) and healing\nprocess (2); and (c) tensile testing of the samples. Table 4 Tensile Strengths of Original and\nRehealed Samples sample σ original (MPa) σ healed (MPa) healing efficiency (η–%) VT-1 105.6 ± 8.1 2.4 ± 0.1 2.3 VT-2 91.9 ± 2.6 7.9 ± 1.6 8.6 VT-3 89.1 ± 3.6 8.6 ± 2.6 9.6 R-VT-1 69.9 ± 2.1 14.6 ± 2.4 20.9 R-VT-2 51.4 ± 1.2 19.3 ± 2.6 37.5 CNT-VT a 56.5 ± 7.2 CNT-VT 95.7 ± 0.4 13.1 ± 1.4 13.7 CNT-RVT 75.4 ± 2.2 17.9 ± 0.7 23.7 a CNT-VT produced without a surfactant. Optimizing the soft-to-hard segment ratio in the epoxy\nresin, R-VT-1\npresents more ductility due to the flexible DGEPG chain segments, Figure 8 . R-VT-1 demonstrates\na 23% decrease in the ultimate tensile strength compared to that of\nVT-2. With increasing soft segment ratio, R-VT-2 displays more ductile\nbehavior along with a lower ultimate tensile strength, Figure 8 . Reinforcing the sample VT-2\nwith CNTs generates 4.1% enhancement in the ultimate tensile strength\ndue to the nanotubes’ individual high mechanical strength 25 , 26 cooperating with the load carrying mechanism. The tensile strength\nof the CNT-RVT sample is increased nearly 8.0% even with a low weight\nratio of CNTs. Similar effects are observed for CNT-reinforced epoxy\nin the literature. 54 , 55 For this reason, introducing\nCNTs is considered an effective approach to compensate the strength\nand toughness for the soft segment-introduced R-VT samples. By this\nmeans, it is aimed to accomplish high healing and mechanical performances\nat the same time. 3.1.8 Morphological Characterization The morphological characteristics of the epoxy networks are investigated\nby scanning electron microscopy (SEM). SEM images are taken at the\ncross-sectional areas with an accelerating voltage of 2 kV under high\nvacuum. The samples are prepared by first breaking them in half followed\nby coating a thin layer of gold. As shown in Figure 9 e, the cross-sectional area of the neat VT\nshows a relatively more smooth fracture surface. Nonetheless, the\nfailure of CNT-VT results in a rougher surface consisting of an epoxy\nlayer and CNT/epoxy layer structure, Figure 9 f. The difference between fracture surfaces\nof the neat VT and CNT-VT is referred to the lower toughness of the\nneat sample. Moreover, the fracture surface of the CNT-VT sample gets\ndistorted due to CNTs causing a more difficult crack propagation. 56 Figure 9 b depicts that even though a surfactant is implemented and\nsonication is performed, CNTs stack more in some regions due to their\nhigh surface energy and hydrophobic surfaces. 57 Such stacking may cause a lower reinforcing effect than the estimated\ntheoretical effect of CNTs in the polymer. 32 Hence, it is crucial to understand the synergistic effect of CNTs\nin the network and their effect on the overall load carrying capability\nof nanocomposites. Figure 9 SEM images of the fracture surfaces of (a) neat VT-2,\n(b) CNT-VT\nwith a surfactant, (c) CNT-RVT with a surfactant, and (d) CNT-VT without\na surfactant. When CNTs are dispersed directly in the resin without\nany functionalization,\nthey tend to agglomerate and terminate with a nonhomogeneous structure\n( Figure 9 d) due to\ntheir high aspect ratio along with van der Waals forces between nanotubes.\nThis leads to a decrease in the mechanical performance, as given in Table 4 , since CNT agglomerates\nbehave as stress concentration points. 48 Contrarily, CNTs on the fracture surface of the samples synthesized\nwith a surfactant, Figure 9 b, appear to be more evenly distributed, promising a more\nefficient load transfer between the nanotube and the matrix. 58 Comparing Figure 9 b,c, the CNT-VT sample is discovered to have a more\nhomogeneous distribution state than that of CNT-RVT. Even though the\ndispersion of CNTs is less than ideal, CNT inclusion is still found\nto enhance the toughness along with a potential increase in healing\nperformance. 3.2 Rehealing After applying the same\nhealing procedure to Ref-Ep-1, Ref-Ep-2, and Ref-R-Ep samples, there\nwas no healing observed at 150° and even 180 °C, justifying\nthe role of disulfide metathesis in the healing mechanism, Figure S9 , wherein VT-2 recovers its original\nstrength of around 9% within disulfide bond exchanges. The healing\nobserved in the VT-2 sample can be misapprehended as the curing of\nthe unreacted species in a stoichiometric imbalance. However, it is\nproven clearly that the healing takes place due to disulfide bond\nexchange considering Ref-Ep-2. The Ref-Ep-2 sample having the same\nstoichiometric imbalance does not show any healing since there are\nno disulfide exchange bonds in the structure, Figure S9 . This signifies that the unreacted species are not\nenough to exhibit healing by themselves. At least three samples\nin each case are studied for healing, and the average values are presented\nin Table 4 . VT-1 shows\nthe lowest healing efficiency, which is associated with the relatively\nlower CAN density and higher crosslink density due to stoichiometry.\nA high crosslink density restrains the stress relaxation in the epoxy\nnetwork and consequently the healing performance. 3 Increased CAN density governs better healing of the broken\nsamples, as VT-2 and VT-3 possess 8.6 and 9.6% healing efficiency,\nrespectively. Moreover, CNT-VT presents 5.1% higher healing efficiency\ncompared to that of the VT-2 sample, as expected from previous considerations. Compared to crosslinked networks, soft segment-introduced samples\npresent higher healing efficiency and relatively lower mechanical\nstrength, as summarized in Table 4 . Increased DGEPG contributes to flexibility and chain\nmobility since the crosslink density decreases proportionally. Owing\nto the increased DGEPG molar ratio, healing efficiency changes remarkably.\nSuch an enhanced healing performance is in good correlation with the\naforementioned relaxation behaviors of VT-2 and R-VT-1 samples. The\nR-VT-1 sample is capable of relaxation even under T g , signifying that exchange reactions begin earlier than T g , Figure 6 a. The R-VT-1 sample shows a 20.9% recovery of the\noriginal strength after healing, wherein the efficiency is 37.5% for\nR-VT-2. It is attributed to the higher flexibility of R-VT-2 since\nhealing favors higher chain mobility and disulfide bond exchange.\nThe increased healing efficiency was forecasted from the heat capacity\nincrease results, Table 3 , having higher motion of molecular chains. In a similar manner,\nCNT-RVT has a healing efficiency of 23.7% having more mobility than\nthat of the R-VT-1 sample. Ultimately, CNT inclusion into R-VT compensates\nthe loss in tensile strength and leads to enhanced healing. Considering\nother rehealable epoxy-related studies in the literature, this study\nprovides a detailed explanation on the effects of both CNTs and soft\nsegments on the healing mechanism simultaneously. 3.3 Reshaping Once the healing behavior\nof dynamic networks is confirmed, reshaping is studied in a hot press.\nSimilar to healing, reshaping is performed not only in dynamic networks\nbut also in reference epoxy samples to point out the importance of\nthe disulfide presence. As can be concluded from Figure S9 , reference epoxy networks cannot be reshaped when\nthere are no reversible disulfide exchange bonds in the structure. As shown in Figure 10 b, epoxy pellets are filled in a circular\ncross-section mold. Soft segment-introduced samples are heated to\n150°C in a hot press and 30 MPa pressure is applied for 1 h.\nThe same procedure is carried out for VT samples at 180 °C. All\nof the epoxy dynamic networks possess reshaping capability due to\ndisulfide bond exchange. The reshaping process is repeated for a second\ntime with the same samples. The resultant samples are in the dog-bone\nshape with a length of 25 mm and a thickness of 2mm. The width is\n10 mm at the tips and 1 mm at the necking region, as shown in Figure 10 d. A tensile test\nis performed using a Deben microtest tensile stage and 200 N load\ncell, and the testing speed is defined as 1.5 mm/min. Figure 11 demonstrates that tensile\nstrengths are reduced for reshaped samples considering their original\nstrengths. Still, the strengths are acceptable, considering that nearly\n45% of the original strength is restored in the CNT-RVT sample after\nreshaping 2 times. A reduction in strength is unavoidable, yet the\nstrengths were still comparable to those of the healed samples. The\nfabricated epoxy dynamic networks are determined to be capable of\nfurther utilization as a component for nonstructural applications. Figure 10 Repeatable\nreshaping process of neat and CNT-reinforced epoxy dynamic\nnetworks; (a) pellets produced from the original sample; (b) reshaped\nsamples—1st cycle; (c) pellets produced from the reshaped sample;\nand (d) reshaped samples—2nd cycle. Figure 11 Tensile strength of 2 times reshaped samples. To verify the properties of the reshaped samples,\nFTIR and DMA\nin the tension mode are conducted, Figures S2 and S8 , respectively. FTIR spectra of the original and reshaped\nsamples are quite similar, remarking that the epoxy network does not\nsuffer from thermal decomposition after the reshaping process. T g values of the reshaped samples are detected\nfrom the peak value of tan δ curves and found to be 124.5\nand 60.7 °C for VT-2 and R-VT-1 samples, respectively. T g values of the reshaped samples are slightly\nhigher than those of the original samples. The storage modulus is\nalso observed to be higher than that of the original samples after\none cycle of reshaping. This is attributed to the shortened macromolecules\nin its structure, which can rearrange easily and densely under applied\nheat and pressure conditions. 59 − 61 It is verified that the reshaping\nprocess is repeatable, although repeated reshaping can generate degradation\ngradually after each time. 61"
} | 8,616 |
23353768 | null | s2 | 6,243 | {
"abstract": "Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium."
} | 169 |
24213281 | PMC3883648 | pmc | 6,244 | {
"abstract": "In eukaryotic cells, cargo is transported on self-organised networks of microtubule trackways by kinesin and dynein motor proteins 1 , 2 . Synthetic microtubule networks have previously been assembled in vitro 3 – 5 and microtubules have been used as shuttles to carry cargoes on lithographically-defined tracks consisting of surface-bound kinesin motors 6 , 7 . Here we show that molecular signals can be used to program both the architecture and the operation of a self-organized transport system based on kinesin and microtubules and spans three orders of magnitude in length scale. A single motor protein - dimeric kinesin 1 8 - is conjugated to various DNA nanostructures to accomplish different tasks. Instructions encoded into the DNA sequences are used to direct the assembly of a polar array of microtubules and can be used to control the loading, active concentration and unloading of cargo on this track network or to trigger the disassembly of the network."
} | 242 |
34632190 | PMC8495691 | pmc | 6,245 | {
"abstract": "CeO 2 was\nsynthesized by the co-precipitation method\non the Cu mesh substrate and modified the surface of CeO 2 @Cu mesh by stearic acid (SA). The superhydrophobic behavior was\nascribed to the combination of hierarchical micro–nanostructure\nof CeO 2 and the hydrophobic alkyl groups from SA. The SA-CeO 2 @Cu mesh had antiacid and base stability and excellent durability\nas well as high separation efficiency. The separation efficiency can\nbe up to 98.0% after separating 30 times.",
"conclusion": "Conclusions To summarize, the superhydrophobic and superoleophilic\nSA-CeO 2 @Cu mesh was prepared with the Cu mesh as the substrate\nand\nSA as the substance with low surface energy. The SA-CeO 2 @Cu mesh had chemical stability (e.g., immersed in acid, base) and\nexcellent durability. The SA-CeO 2 @Cu mesh can separate\ndifferent oils that are insoluble in water and had a separation efficiency\nabove 98% after 30 separation cycles. The excellent reusability and\nthe high separation efficiency result in the possibility of the practical\napplication of the SA-CeO 2 @Cu mesh for oil–water\nseparation.",
"introduction": "Introduction It is pretty difficult to treat the increasing\nemissions of industrial\noily wastewater because of the pressure of environmental protection\nand ecological balance. Therefore, the treatment of oily wastewater\nis one of the urgent problems in the field of environmental engineering.\nThe traditional methods, such as flotation method, 1 , 2 flocculation\nmethod, 3 etc., have been unable to meet\nthe current environmental requirements, and it is imperative to adopt\nnew, efficient, and functional oil absorption or oil–water\nseparation materials. Superhydrophobic and superhydrophilic materials 4 − 7 have received great attention due to their potential application\nin oil–water separation. Only oil can pass through these materials\ndue to their superhydrophilicity, while water is totally repelled\ndue to their superhydrophobicity. Superhydrophobic–superhydrophilic\nsurface has unique advantages, such as self-cleaning effect, 8 , 9 superhydrophobic materials have properties of antifouling, 10 , 11 anticorrosion, 12 , 13 anti-icing, 14 , 15 drag reduction, 16 , 17 etc. Due to the large difference\nin surface tension between the oil phase and the water phase in the\noil–water mixture, superhydrophobic materials can be used as\nthe difference to selectively remove water or oil, thereby achieving\nefficient oil–water separation. Many artificial superhydrophobic\nmembrane materials, such as metallic\nmeshes, 18 , 19 sponge, 20 , 21 and ceramic\nmaterials, 22 , 23 etc, have become the focus of\nresearch on oil–water separation materials due to their high\nflux, low cost, simple operation, and high processing efficiency. As we know, proper roughness and low surface energy are the key\nfactors to prepare superhydrophobic surfaces. 24 − 26 The proper\nroughness is useful to improve the performance of filtration membranes.\nThe relationship between surface roughness and water repellency was\nworked out by Cassie and Baxter 27 as well\nas Wenzel. 28 Various methods are used to\nfabricate superhydrophobic surfaces, such as self-assembly 29 , 30 electrospinning, 31 sol–gel methods, 32 etc. Many researchers have studied the practical\napplications of superhydrophobic materials. However, the poor stability\nand reusability of superhydrophobic materials seriously hindered their\npractical applications. It is urgently necessary to design superhydrophobic\nmaterials that have low cost and high separation efficiency and are\nenvironmentally friendly for oil–water separation. The membrane\nseparation method is widely used in the treatment of oily wastewater\nbecause of its simple operation, good treatment effect, no secondary\npollution, and low-energy consumption. Recently, Azimi et al.\nreported that rare-earth oxide (REO) surfaces\nshowed their thermally stable hydrophobicity after exposure to 1000\n°C. 33 Moreover, Azimi et al. also\nrevealed that REOs become superhydrophobic with textured morphology.\nCompared with organic metals, these inorganic REOs are thermally and\nmechanically more stable, which allows us to use them as a novel durable\nhydrophobic coating. Zenkin et al. 34 prepared\nNd 2 O 3 , La 2 O 3 , and Y 2 O 3 by the sputtering method and proved that the\nhydrophobicity was related to the nonpolar component of the surfaces\nof REOs. CeO 2 is an inexpensive and widely used rare-earth\ncompound with N-type semiconductor properties and a unique 4f electronic\nstructure. It exhibits excellent oxygen storage and charge exchange\ncapacity and is widely used in three-way catalysts, photocatalysis,\nwastewater treatment, electronic ceramics, etc. As a surface modifier,\nSA has nontoxic biocompatibility, and it is not easy to change the\nphysical and chemical properties of the powder. The surfaces of metallic\nmeshes have been modified by low-energy materials or nanoparticles\nto form superhydrophobic and superoleophilic for oil–water\nseparation. According to the different mesh shapes and densities,\nthe function of the copper mesh is different. The main role of copper\nmesh is screening, filtration, protection, etc. However, as far as\nwe know, no works have reported the SA-CeO 2 @Cu mesh with\nsuperhydrophobic and superoleophilic properties for oil–water\nseparation. Herein, we report a superhydrophobic and superoleophilic\nCeO 2 -coated Cu mesh by the co-precipitation method and\ncalcination\nand then used SA as a low-energy material. In addition, the SA-CeO 2 @Cu mesh showed desirable stability and high oil–water\nseparation efficiency when suffered from acid and base. The prepared\nSA-CeO 2 @Cu mesh was also characterized. The oil–water\nseparation experiments and the recyclability of SA-CeO 2 @Cu mesh were also investigated.",
"discussion": "Results and Discussion X-ray\nDiffraction (XRD) Analysis As shown in Figure 1 a,b, the XRD pattern\nof CeO 2 and SA-CeO 2 were consistent with the\nvalues in the standard card (JCPDS No. 34-0394). The diffraction peaks\nobserved at 2θ values of 27.28, 33.58, 46.46, 57.08, 59.26,\n68.88, 76.48, and 78.82° can be assigned to the (111), (200),\n(220), (311), (222), (400), (331), and (420) crystal planes of fluorite,\nrespectively. The XRD pattern of SA-CeO 2 was similar to\nthat of CeO 2 without any peaks attributed to the SA molecules,\nindicating that the crystalline phase of CeO 2 did not change\nafter the treatment with SA. Figure 1 Image of XRD pattern of (a) CeO 2 and\n(b) SA-CeO 2 . FT-IR Analysis As shown in Figure 2 a, the broad peak around 3400 cm –1 was the bond\nstretching vibration absorption peak and the characteristic\npeak of the crystal water, which indicated that the sample contains\ncrystal water. The surface modification process of CeO 2 was a process in which the carboxyl group in SA reacted with the\nhydroxyl group on the surface of CeO 2 to change the surface\nproperties of CeO 2 . Compared with the unmodified CeO 2 , the broad peak of SA-CeO 2 around 3400 cm –1 was the disappearance of the −OH stretching\nvibration absorption peak, indicating that the carboxyl group in SA\nwas chemically bonded to the hydroxyl group on the surface of CeO 2 . As shown in Figure 2 b, the characteristic absorption peak at 2924 and 2858 cm –1 were the −C–H stretching vibration\npeaks in −CH 2 and −CH 3 . The carboxyl\nstretching vibration peak appeared at 1710 cm –1 ,\nwhich was consistent with the fatty long carbon chain group in SA,\nwhich indicated the presence of SA on the surface of the modified\nCeO 2 . Figure 2 FT-IR spectrum of (a) CeO 2 and (b) SA-CeO 2 . Morphological Analysis\nof the CeO 2 @Cu Mesh As shown in Figure 3 , the microscopic morphology\nanalysis of the surface of the untreated\nCu mesh and the surface of the CeO 2 @Cu mesh. As shown in Figure 3 a, the surface looked\nsmooth at low magnification. After calcination, a uniform rod-shaped\nCeO 2 with a size of about 100 nm was immobilized on the\nsurface of CeO 2 @Cu mesh, which provided a nanolevel roughness\nto the surface of the Cu mesh. Also, the CeO 2 nanoparticles\nexhibit immobilization with a resistance sonication in ethanol for\n30 min with a significant amount of CeO 2 nanoparticles\nstill anchored on the Cu mesh surface. The CeO 2 @Cu mesh\nhad a micro–nano-level layered structure, in which CeO 2 nanoparticles were fixed on the surface of the copper mesh\nwire, which was the key factor for superhydrophobicity. 35 The CeO 2 nanoparticles can effectively\nreduce the contact area between the water drops and Cu mesh. Figure 3 SEM images\nof (a) the untreated Cu mesh and (b)–(d) CeO 2 @Cu\nmesh. Wettability Tests and Adhesion\nPerformance Analysis As shown in Figure 4 a, the CA of water droplets on the untreated\ncopper mesh was 82.1\n± 2°, and the oil droplets would spread out quickly on the\nuntreated copper mesh. As shown in Figure 4 b–d, the CA of water droplets on the\nCeO 2 @Cu mesh was 137.5 ± 2°, and the CAs of the\nSA-CeO 2 @Cu mesh immersed in SA for 1 h and 12 h were 150.1\n± 2° and 158.1 ± 2°, respectively. Due to the\ncapillary effect produced by the microstructure, water was prevented\nfrom passing through the SA-CeO 2 @Cu mesh. The micro-/nanostructure\ncan reduce the contact area between the solid–liquid and water\nand low surface energy SA, which reacted with the Cu mesh to generate\ncopper stearate. The SA-CeO 2 @Cu mesh showed low adhesion\nto water. Figure 4 CA on different Cu mesh surfaces water droplets on (a) untreated\nCu mesh, (b) CeO 2 @Cu mesh, (c) CeO 2 @Cu mesh\nfor 1 h, and (d) CeO 2 @Cu mesh for 12 h. To have a better understanding of the superhydrophobic behavior\nof the SA-CeO 2 @Cu mesh, the wetting state and the oil–water\nseparation performance were studied. Due to the modification of the\nlow-energy material SA together with CeO 2 nanoparticles,\nwater droplets would be in the superhydrophobic Cassie state and high\nWCA can be explained by the Cassie–Baxter equation 27 , 36 as follows. 1 where θ 2 is the WCA on the\nSA-CeO 2 @Cu mesh, θ 1 is the WCA on the\noriginal Cu mesh, and f 1 and f 2 ( f 1 + f 2 = 1) are the area frictions of water connecting with\nthe SA-CeO 2 @Cu mesh and air, respectively. According to eq 2 , f 2 can be calculated to be 0.937, indicating that 93.7% surface\narea was covered by air, which resulted in the superhydrophobicity\nof the SA-CeO 2 @Cu mesh. According to the Wenzel equation, 28 the superoleophilic properties can be enhanced\nby the nanostructures. Therefore, when placed on the SA-CeO 2 @Cu mesh, the oil droplet would enter the nanostructures due to the\ncapillary effect. The combination of the hydrophobic surface chemistry\nand roughness would lead to superhydrophobic and superoleophilic properties\neasily. As shown in the low adhesion image of water droplets\nand the SA-CeO 2 @Cu mesh in Figure 5 , 5 μL water droplets were suspended\non the needle tube.\nBy controlling the experimental platform, the water droplets contacted\nwith the surface of the SA-CeO 2 @Cu mesh. Continuous rise\nin the experimental platform caused the water droplets and the surface\nof the SA-CeO 2 @Cu mesh to squeeze; then, as the experimental\nplatform descended, the water droplets and the surface of the SA-CeO 2 @Cu mesh completely separated. Figure 5 Adhesion images of the\nwater droplets on the SA-CeO 2 @Cu mesh. When the SA-CeO 2 @Cu mesh and the water droplets were\ncompletely separated, the water droplets recovered their original\nappearance and were fully suspended on the needle tube, and no liquid\nwas adhered to the surface of the SA-CeO 2 @Cu mesh, indicating\nthat the prepared SA-CeO 2 @Cu mesh surface had low adhesion\nto water droplets, and low adhesion performance was very important\nfor superhydrophobic surfaces in the practical application of self-cleaning. Oil–Water Separation Toluene, cyclohexane, and\nkerosene were chosen as the oil phases. The separation efficiency\nof different oil–water mixtures when the mass of the oil phase\nwas 10.0 g and the oil–water mass ratio was 1:1 is shown in Figure 6 . Figure 6 Separation efficiency\nwith different separation times. The separation efficiency of SA-CeO 2 @Cu mesh was calculated\nby eq 2 . For the oil–water\nmixture with a mass ratio of 1:1, the first separation efficiency\nwas as high as 99%. After several separation cycles, the separation\nefficiency can reach more than 98%, indicating that the SA-CeO 2 @Cu mesh still maintained the water–oil separation\nability. In addition, the SA-CeO 2 @Cu mesh can separate\ndifferent oil–water mixtures (water–cyclohexane, water–toluene,\nwater–kerosene), indicating that the SA-CeO 2 @Cu\nmesh can generally separate oil–water mixture. After 30 separation\ncycles, the oil–water separation efficiency of the SA-CeO 2 @Cu mesh is still above 98%, indicating that it can be used\nas a filmlike material to efficiently separate an oil–water\nmixture, and the mechanical stability of the Cu mesh was excellent. Chemical Stability Chemical stability is important\nfor practical applications. Therefore, to examine its chemical stability,\nthe SA-CeO 2 @Cu mesh was immersed into an acid solution\n(HCl, pH = 1) and a base solution (NaOH, pH = 12) for 72 h; then,\nthe wettabilities and oil–water separation experiment of the\nSA-CeO 2 @Cu mesh were investigated. As shown in Figure 7 , after immersing\nfor 72 h, the CA in the hydrochloric acid solution with pH 1 was 156.3\n± 2° and that in the sodium hydroxide solution with pH 12\nwas 156.4 ± 2°; the oil–water separation efficiency\nremained above 98%, indicating that the SA-CeO 2 @Cu mesh\nstill maintained superhydrophobicity, which proved the excellent acid\nand alkali resistance of the SA-CeO 2 @Cu mesh. Figure 7 (a) CA at different\nimmersion times. (b) Separation efficiency\nat different immersion times."
} | 3,427 |
28834251 | PMC5609238 | pmc | 6,248 | {
"abstract": "Summary Microbial biotechnology is essential for the development of circular economy in wastewater treatment by integrating energy production and resource recovery into the production of clean water. A comprehensive knowledge about identity, physiology, ecology, and population dynamics of process‐critical microorganisms will improve process stability, reduce CO2 footprints, optimize recovery and bioenergy production, and help finding new approaches and solutions. Examples of research needs and perspectives are provided, demonstrating the great importance of microbial biotechnology."
} | 147 |
34754966 | PMC8554343 | pmc | 6,249 | {
"abstract": "Cyanobacteria can utilize CO 2 or even N 2 to produce a variety of high value-added products efficiently. Plastoquinone (PQ) is an important electron carrier in both of the photosynthetic and respiratory electron transport chain. Although the content of PQ, as well as their redox state, have an important effect on physiology and metabolism, there are relatively few studies on the synthesis of PQ and its related metabolic regulation mechanism in photosynthetic microorganisms. In this study, the strategies of overexpression of Geranyl diphosphate: 4-hydroxybenzoate geranyltransferase ( lepgt ) and addition of 4-hydroxybenzoate (4-HB) as the quinone ring precursor were adopted to regulate the biosynthesis of PQ in Synechocystis PCC 6803. Combined with the analysis the photosystem activity, respiration rate and metabolic components, we found the changes of intracellular PQ reprogrammed the metabolism of Synechocystis PCC 6803. The results showed that the overexpression of lepgt reduced PQ content dramatically, by 22.18%. Interestingly, both of the photosynthesis and respiration rate were enhanced. In addition, the intracellular lipid and protein contents were significantly increased. Whereas, the addition of low concentrations of 4-HB enhanced the biosynthesis of PQ, and the intracellular PQ contents were increased by 14.76%–70.86% in different conditions. Addition of 4-HB can regulate the photosystem efficiency and respiration and reprogram the metabolism of Synechocystis PCC 6803 efficiently. In a word, regulating the PQ biosynthesis provided a novel idea for promoting the reprogramming the physiology and metabolism of Synechocystis .",
"conclusion": "4 Conclusion PQ is an important redox cofactor in Cyanobacterium. In this study, the synthesis of PQ in Synechocystis were regulated by two strategies: genetic engineering and the addition of 4-HB. The overexpression of lepgt and the addition of low-concentration 4-HB had little effect on the growth and promoted the synthesis of neutral lipid. Small changes in PQ content would lead to more changes of metabolic flux and result in different phenotypes. This study explored the effect of PQ content changes on the metabolic regulation of prokaryotic photoautotrophs. The strategies of genetic engineering and compound addition in this work provide a new method for regulating the anabolism of active substances in cyanobacteria.",
"introduction": "1 Introduction Cyanobacteria is a photosynthetic microorganism, which can use CO 2 even N 2 to efficiently synthesize high value-added compounds (e.g. ethanol, fatty acids, isoprene) [ 1 ]. It has broad application prospects in the fields of food, bioenergy and environments. A variety of strategies, such as genetic engineering or stress tolerance, have been applied to increasing the level of target products of Cyanobacteria. For example, both nitrogen stress conditions [ 2 ] and overexpression of key enzymes [ 3 ] can be used to increase lipids accumulation. Reducing power and energy play a crucial role in the growth and metabolism of algal cells. The change and consumption rate of NADPH and ATP in algae can regulate the intracellular physiological metabolic rate and biomass productivity [ [4] , [5] , [6] ]. Synechocystis is one of the most widely used prokaryotic model cyanobacteria nowadays [ 7 ]. Synechocystis sp. PCC 6803 is a model cyanobacteria which can synthesize glycogen, fatty acids and polyhydroxybutyrate. Photosynthetic and respiratory electron transport chain plays a vital role in photosynthetic microorganisms. Plastoquinone (PQ) is an important electron transfer intermediate, which mediates the transfer of reducing power and energy in Synechocystis [ 8 ]. In Synechocystis , the quinone ring precursor of PQ is 4-hydroxybenzoic acid (4-HB), which is different from eukaryotic microalgae [ 9 ]. The synthesis pathway of 4-HB in Synechocystis is similar to that of Escherichia coli which synthesized from chorismite [ 10 ]. The isoprene side chain precursor of PQ is solanesyl diphosphate (SPP) or dipropyl diphosphate (DPS), which is synthesized through the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in Synechocystis [ 11 ]. In the second synthesis stage, 4-HB and SPP undergo a series of decarboxylation, hydroxylation, methylation, and other steps to synthesize PQ-9. However, the genes and metabolic pathways of the PQ biosynthetic pathway in Synechocystis are not completely understood yet. The content and the redox status of PQ are important to the growth and metabolism of cyanobacteria. Firstly, PQ acts as a carrier of electrons in the photosynthetic electron transport chain. PQ is an intermediate of acyclic electron transport chain, which transmits electrons from PSII to cytochrome b 6 f (Cyt b 6 f ). Besides, PQ is also an important part of the cyclic electron transport chain. In this cycle, electrons can be transmitted from PQ to PSI through Cyt b 6 f , then back to PQ through reduced ferredoxin or NADPH [ 12 ]. What's more, PQ also plays an important role in respiratory electron transport chain in Synechocystis . There are many studies on the PQ-mediated photosynthetic electron transport chain, while relatively few exploration on the PQ-mediated respiratory electron transport chain of Synechocystis . It is known that the electrons generated by the substrate on the respiratory electron transport chain are transferred to PQ through NDH-1 or SDH, and then transferred to electron carriers such as cyt b 6 f , and finally transferred to terminal oxidase [ 13 ]. Studies have shown that electrons generated from respiratory can participate in the photosynthetic electron transport chain too [ 14 ]. The respiratory chain can provide or remove electrons in the photosynthetic electron transport chain to prevent excessive oxidation or reduction of PQ [ 15 ]. The energy and reducing power generated by the PQ-mediated photosynthetic and respiratory electron transport chain can participate in the synthesis of various important chemical compounds in Synechocystis . Furthermore, PQ is involved in the anabolism of various compounds in microalgae and cyanobacteria. For example, it is involved in the synthesis of carotenoids [ 16 ]. It plays an important role in the formation of the transmembrane proton gradient required for ATP synthesis in the chloroplast [ 17 ]. Finally, PQ can participate in a variety of biological behaviors, including acting as redox sensor [ 18 ], responding to heavy metal ion concentration changes [ 19 ], responding to biological pressure [ 20 ], and mediating programmed death [ 21 ]. As a carrier of electrons for photosynthesis and respiration, changes in PQ content will have a vital impact on the growth and metabolism of photosynthetic organisms [ 22 ]. Previous studies have found that complete lack of PQ would result corn to turn yellow and was prone to death in the seedling stage [ 23 ]. The complete lack of PQ inhibits the photosynthetic electron transfer in the chloroplast and results in overexcitation of PSII and PSI [ 24 ]. Excessive excitation of PSII and PSI will produce a large amount of reactive oxygen species (ROS), cause severe photo-oxidation damage, and seriously affect chloroplast metabolism [ 25 ]. The complete lack of PQ in the mesophyll cell (MC) results in the accumulation of large starch granules in the chloroplasts, but no starch accumulation in bundle sheath cells (BSC) chloroplasts of maize [ 26 ]. The difference in the degradation rate of ROS between MC and BSC chloroplasts indicates that there are differences in the mechanism of physiological metabolism and synthesis between the two organelle types. Therefore, the variation of PQ content in different photosynthetic organisms may lead to different phenotypes. Geranyl diphosphate: 4-hydroxybenzoate geranyltransferase ( lepgt ) from Lithospermum erythrorhizon is a key catalytic enzyme in shikonin biosynthesis pathway. LePGT synthesizes isoprene 4-hydroxybenzoate (G-4HB) by using 4-HB and GPP [ 27 , 28 ]. Interestingly, 4-HB and GPP are the same substrates for ubiquinone biosynthesis [ 29 ]. We previously studied the expression of lepgt in E. coli , and found that ubiquinone (UQ) content was reduced and led to the accumulation of lactic acid, which was approximately the theoretical yield [ 30 ]. It was proved that small changes in UQ content in E. coli could achieve precise regulation of metabolism. In addition, green microalgae, such as Chlamydomonas reinhardtii , can synthesize UQ by adding 4-HB [ 31 ]. Here, we attempted to regulate the intracellular PQ content through the strategies of overexpressing lepgt or adding 4-HB to Synechocystis . The metabolic rules like photosystem efficiency and respiration rate, as well as intracellular metabolite production mode were studied. To the best of our knowledge, this is the first attempt to discuss the relationship between PQ content and intracellular metabolites in photoautotrophic microorganism.",
"discussion": "3 Results and discussion 3.1 Two strategies of regulating the PQ biosynthesis in Synechocystis To reduce the content of PQ, we constructed the plasmid pUC- lepgt and integrated the expression cassette of lepgt into the Synechocystis chromosome through natural transformation and homologous recombination ( Fig. 1 a). The single colony, after multiple purification, was boiling to quickly extract the genome. We verified the upstream homology arms, the target gene and the downstream homology arm by using primers Up-F, psbAII -R, primers LePGT -F and LePGT -R and Trbcl -F and down-R, respectively ( Table S1 and Fig. 1 b). The successfully integrated single algal colonies were selected and cultured to the middle of logarithmic growth phase. RNA was extracted and reverse transcribed into cDNA ( Fig. 1 c). We confirmed that the lepgt in the selected transformants was successfully transcribed by identifying the DNA and RNA of the transformants. Phenotypes of transformants and wild type strains were measured. The content of PQ in transformant was significantly reduced by 22.18% ( p < 0.05) compared with the wild-type ( Fig. 1 d). In another strategy, different concentrations (0 mM, 1 mM, 2 mM) of 4-hydroxybenzoic acid (4-HB) were added to promote the synthesis of PQ in Synechocystis . The experimental group of 0 mM was wild type Synechocystis with 1% (v/v) ethanol. After addition of 1 mM 4-HB, the intracellular PQ content of the experimental group increased by 14.76% than that of the control. In addition, when Synechocystis cultured in the medium containing 2 mM 4-HB, the intracellular PQ content increased to 596.16 nmol/OD and significantly increased by 70.86% than that of the control one ( p < 0.01) ( Fig. 1 d). Different from eukaryotic microalgae, the quinone ring precursor of PQ in Synechocystis is 4-HB and the precursor of isoprene side chain is GPP. Therefore, the overexpression of lepgt in Synechocystis can drain the precursors of PQ, 4-HB and GPP, to reduce PQ content. The increasing of PQ content with 4-HB treatment confirmed that Synechocystis could synthesize PQ with exogenous 4-HB. The changes of PQ content will affect electron transfer efficiency of the photosynthetic and respiratory, and further affect the efficiency of photosynthetic and respiratory system. It ultimately influences production of reducing power and energy. Hence, small changes in PQ content will probably reprogram the intracellular metabolic flux. 3.2 Changes in PQ content had little effect on the growth The growth curves of the wild type and the transformant showed no significant difference ( Fig. 1 e). The results indicated that the small amount of reduced PQ content might have no significant effect on the growth of Synechocystis . Meanwhile, the effect of the addition of 4-HB on the growth rate of Synechocystis were also studied. As shown in Fig. 1 e, compared to the control group, the addition of 1 mM 4-HB and 2 mM 4-HB had little effect on the growth of Synechocystis . However, higher concentrations (3 mM) inhibited the growth of Synechocystis (50% at 240 h), and the growth was completely inhibited with 5 mM addition (data not shown). In order to further study the effects on metabolism of Synechocystis , we measured the efficiency of the photosystem, respiration rate, and the concentrations of intracellular metabolites in Synechocystis . 3.3 The effect of changes in PQ content on photosystem II 3.3.1 The effect of reduced PQ content on photosystem II In order to further study the effect of PQ content reduction on photosynthesis, the chlorophyll fluorescence parameters of the wild type and transformant in the exponential phase were measured and analyzed according to previous method [ 36 ]. The fluorescence values at step O, J, and I (Fo, Fj, Fi) and the maximum fluorescence value (Fm) of the transformant were all higher than those of the wild type ( Table S2 ). The basic shape of the OJIP fast fluorescence kinetic curve was consistent with that of the wild type ( Fig. 2 a and c). The initial slope of the fluorescence curve (Mo) and the fluorescence level of step J (Vj) decreased by 7.95% and 7.55%, respectively. This suggested that overexpression of lepgt promoted the electron transport chain between Q a and Q b in Synechocystis [ 37 ]. The values of parameter Sm and Ψ0 in transformant were both higher than those of the wild type ( Table S2 ). It was indicated that the transfer yield of per captured electron in transformant was higher [ 38 ]. Though the values of Fv and Fm in transformant were increased by 9.32% and 17.62%, respectively, the maximum electron transfer efficiency (Fv/Fm) of the photosystem II (PSII) of transformant was slightly increased by 0.24%. Fig. 2 Chlorophyll a fluorescence transients of Synechocystis PCC 6803. (a): Chlorophyll a fluorescence kinetics curve of wild-type and transformant; (b): Chlorophyll a fluorescence kinetics curve with different concentrations of 4-HB; (c): The spider-plot presentation of selected parameters quantifying the behavior of PS II of wild-type and transformant; (d): Spider-plot presentation of selected parameters quantifying the behavior of PS II with different concentrations of 4-HB. The group of 0 mM was wild type Synechocystis which 1% ethanol was added. Fig. 2 PI ABS , based on the “vitality” index or survival index of light quantum flux absorption, was the overall expression of photosynthetic activity. The PI ABS value of the transformant increased by 22.54% which proved that the overexpression of lepgt increased the “vitality” of PSII in Synechocystis . The values of φ Po and φ Eo of the transformant increased by 12.94% and 9.21% than the wild-type, respectively. The overexpression of lepgt enhanced the capture efficiency of absorbed energy and the transfer the absorbed energy to the photosynthetic electron transport chain. When we considered the changes in the specific energy fluxes used for energy absorption (ABS/RC), capture (TRo/RC), transport (ETo/RC), and consumption (DIo/RC), the data showed that the values of absorption and capture efficiency were slightly reduced and the electron transfer efficiency was increased by 11.55% compared to the wild type. This indicated that the overexpression of lepgt might increase the electron transfer efficiency and reduce the energy dissipation efficiency in Synechocystis . We found that the PI ABS value of the transformant increased due to the increasing in the efficiency of electron transport in the photosynthetic pathway and other pathways flowing into the photosynthetic electron transport chain [ 39 ]. qPQ represented the non-photochemical quenching of PSII and the value of qPQ in transformant significantly increased by 180.70% ( p < 0.05). It was proved that the energy absorption in the photosynthetic electron transport chain of the transformant was much higher than the energy utilization efficiency and the over-excitation of PSII was prevented by non-photochemical quenching. 3.3.2 The effect of different increments of PQ on photosystem II We measured the effect on the chlorophyll fluorescence parameters of Synechocystis with low-concentration 4-HB addition. The results showed that the initial slope of the fluorescence curve (Mo) and the fluorescence level of step J (Vj) gradually increased with the increasing of 4-HB concentration ( Fig. 2 b and d). Compared with the control group, the values of Mo, V i and Vj amounted to 149.20%, 193.65%, and 152.16% of the control in the Synechocystis treated with 2 mM 4-HB. The addition of low-concentration 4-HB inhibited electron transfer from Q a to Q b in Synechocystis [ 40 ]. The basic shape of the OJIP fast fluorescence kinetic curve of Synechocystis with 1 mM 4-HB addition was consistent with that of the control group, but it changed with 2 mM 4-HB addition. Though the values of the parameter Fv and Fm of the experimental group treated with 1 mM 4-HB were diminished by 11.30% and 9.26%, respectively, the maximum electron transfer efficiency (Fv/Fm) of PSII slightly reduced and had no significant change compared with the control group. The values of the Fv and Fm at 2 mM of 4-HB were dropped by 70.48% and 70.09%, respectively. The value of Fv/Fm at 2 mM of 4-HB was only 0.04 and significantly decreased by 83.82% ( p < 0.05). 4-HB diminished the Fv/Fm value of Synechocystis in a concentration-dependent manner [ 41 ]. We found that the PI ABS value of Synechocystis gradually decreased with the increasing of 4-HB concentration. The PI ABS value with 2 mM 4-HB addition was only 0.015. This result showed that the addition of 2 mM 4-HB could significantly decrease “vitality” of photosynthesis of Synechocystis . N is the reduction turnover amount of PQ. The N value of Synechocystis with 2 mM 4-HB improved by 494.36% which proved that 4-HB improved turnover numbers of PQ. The values of parameter ETo/RC, ETo/ABS and ETo/TRo in Synechocystis treated with 2 mM 4-HB dropped by 33.86%, 20.83% and 9.60%, respectively, and the DI O /RC value increased by 974.86% compared with the control group. This indicated that the addition of 2 mM 4-HB could reduce the transfer efficiency of absorbed energy and captured energy and the electron transfer of per unit reaction center. It also increased the efficiency of energy dissipation in Synechocystis . In summary, by measuring the chlorophyll fluorescence parameters, we found that the PSII efficiency with 1 mM 4-HB addition was slightly reduced, whereas was significantly reduced with 2 mM 4-HB addition. The decrease of PI ABS value in Synechocystis with 4-HB addition was diminished, because of the reducing of electron transfer rate in the electron transport chain and the increasing of energy dissipation efficiency [ 40 ]. 3.4 The effect of changes in PQ content on photosystem I 3.4.1 The effect of reduced PQ content on photosystem I In the photosynthesis of Synechocystis , Photosystem I (PSI) could transfer electrons from phycocyanin or cytochrome to ferredoxin. PSI is the most efficient photoelectric device in nature which every photon captured could be used for electron displacement [ 42 ]. Photosynthetic cyclic electron transport is the photosynthetic electron transport around PSI which starts and ends with PSI. It only produces ATP and does not produce NADPH which could regulate the ratio of ATP/NADPH [ 43 ]. As a carrier of electrons for photosynthesis and respiration, changes of PQ content will have a vital impact on the photosynthesis and respiration efficiency of photosynthetic organisms [ 44 ]. Under the stimulation of far red light and white light, the total photosystem efficiency of PSI in Synechocystis was measured by Dual-PAM-100. The parameter of P700 represents the photosynthetic system efficiency. The parameter of P700 + represents the efficiency of the linear electron transfer chain and the amount of PSI [ 45 ]. We found that the total photosystem efficiency of transformant was higher than the wild type ( Fig. 3 a and b). Our results indicated that the expression of lepgt increased the total photosystem efficiency of Synechocystis . DCMU could inhibit the electron transport from PSII to PQ, thus blocking the linear electron transport chain. With the addition of DCMU and far red light, we measured the efficiency of cyclic electron transport (P700 + ) in S ynechocystis and found the photosystem I efficiency of the transformant in transformant was increased by 40.51% compared with the wild type ( Fig. 3 b). Taken together, the overexpression of lepgt increased the amount of PSI in the transformants. In conclusion, the overexpression of lepgt in Synechocystis diminished the PQ content, promoted the linear electron transport and the total photosystem efficiency of photosynthesis, and had little effect on the efficiency of cyclic electron transport chain. It would benefit to the NADPH and ATP produced by photosynthesis and regulated the NADPH/ATP ratio produced by photosynthesis. Fig. 3 Phenotypes variation of photosystem I and respiration of Synechocystis PCC 6803. (a): Effect of the overexpressed lepgt and exogenous 4-HB on the intital rate of 700 dark reduction; (b): Effect of the overexpression of lepgt and exogenous addition of 4-HB on the intital rate of P700 + dark reduction; (c): Respiration rate of Synechocystis PCC 6803. The group of 0 mM was wild type Synechocystis which 1% ethanol was added. The value obtained for WT was considered as 100. Standard errors was calculated from three biological independent experiments; asterisks represent significant differences (* p < 0.05, ** p < 0.01). Fig. 3 3.4.2 The effect of different increments of PQ on photosystem I The total photosystem efficiency in Synechocystis with low-concentration 4-HB was measured by Dual-PAM-100 with the stimulation of far red light and white light. The results showed that 4-HB can effectively improve the total photosystem efficiency (P700) at 1 mM addition and it can significantly inhibit photosystem at 2 mM addition ( Fig. 3 a). Then, we measured the photosystem I efficiency with the addition of DCMU and far red light ( Fig. 3 b) [ 45 ]. The results showed that 4-HB slightly increased the PSI efficiency of Synechocystis at 1 mM addition. However, with higher concentrations (2 mM) 4-HB showed inhibition effect. Furthermore, the number of PSI with 1 mM 4-HB addition slightly increased by 13.87% while the number of PSI treated with 2 mM 4-HB dropped by 50.82% in cyanobacteria. In conclusion, 4-HB could slightly decrease the efficiency of PSII and increase the efficiency of cycle electron transport and the total photosystem at 1 mM. We assumed that the addition of 1 mM 4-HB slightly decreased the NADPH content produced by the linear electron transport chain, increased the ATP content produced by the cycle electron transport chain and adjusted the ratio of NADPH/ATP produced by the photosystem in Synechocystis . However, higher concentrations (2 mM) significantly restrain the efficiency of photosystem and decrease the production of both NADPH and ATP. Detailed multi-omics analysis is need to be conducted to further elucidate the mechanisms beyond the PQ regulation. 3.5 The effect of changes in PQ content on respiration rate Respiration in cyanobacteria maintains a transmembrane proton gradient in the dark to promote the production of ATP and water, thus provides energy for some active transport [ 46 ]. In addition, PQ mediated respiration and photosynthesis are both on the thylakoid membrane. Therefore, the data of photosynthesis and respiratory system should be combined to analyze the impact of PQ content fluctuation on Synechocystis . We used FirestingGO 2 oxygen electrode to measure the respiration rates of Synechocystis in the logarithmic period under dark conditions. As shown in Fig. 3 c, the respiration rate of the transformant increased by 27.35% compared to the wild-type. In order to prevent the photosystem from overreduction, the metabolic cooperation between photosynthetic and respiratory is needed to supply more ATP [ 47 ]. From these results, we could infer that the overexpression of lepgt in Synechocystis could significantly reduce PQ content and significantly increase photochemical quenching efficiency of the photosystem. Therefore, the respiration rate was enhanced to promote the production of ATP. As shown in Fig. 3 c, the respiration rate of control group amounted to 20.20 μmol O 2 /mg Chl/h and increased by 20.01% compared with WT. It was an interesting phenomenon that the addition of 1% ethanol decreased the efficiency of total photosystem and improved the respiration rate. The respiration rate of cells with 1 mM 4-HB addition increased by 20.08%, and the respiration rate of cells with 2 mM 4-HB addition reduced by 54.90% ( p < 0.01). Combined with the analysis of chlorophyll fluorescence parameters, we found that the addition of 1 mM 4-HB had almost no significant effect on the PSII efficiency and increased the total photosystem efficiency and respiration rate. However, with the addition of 2 mM 4-HB, both photosystem efficiency and respiratory rate reduced significantly. Although the addition of two different concentrations of 4-HB both can lead to the increase of PQ content, it will have completely different effects on photosynthesis and respiration. This indicated that PQ not only acts as redox intermediate, but also affects the physiological state of Synechocystis in other ways. Thus the changes of PQ content will have a vital impact on the photosynthesis and respiration of Cyanobacteria. 3.6 The effect of changes in PQ content on intracellular metabolites 3.6.1 The effect of reduced PQ content on intracellular metabolites Lipid and glycogen are the main intracellular reduction products of Synechocystis . Glycogen is the main carbohydrates store in cyanobacteria and has a wide range of biological functions. For example, it can be converted to bioethanol by yeast fermentation or be converted to methane by anaerobic fermentation in biogas plants [ 48 ]. Glycogen production in cyanobacteria can be significantly promoted under nitrogen stress. However, the nutritional stress significantly reduced the growth [ 49 ]. The overexpression of lepgt decreased the content of PQ and promoted the efficiency of photosynthesis and respiration in Synechocystis . In order to study the regulation mechanism of reduced PQ content on intracellular metabolism, the changes of fatty acids, glycogen, protein and other metabolites were analyzed ( Table 1 ). Table 1 Physiological metabolism parameters of Synechocystis PCC 6803 with different treatments. Table 1 WT LePGT Control 1 mM 4-HB 2 mM 4-HB Caretoind (μg/mL) 2.27 ± 0.31 2.38 ± 0.050 2.03 ± 0.085 1.91 ± 0.015 1.86 ± 0.025 PQ (nmol/OD730) 337.78 ± 11.64 262.86* ± 6.47 348.91 ± 16.34 400.40 ± 15.81 596.16 ## ± 21.43 Glycogen (mg/DCW) 170.15 ± 15.73 173.76 ± 14.21 155.89 ± 3.93 205.21 ## ± 15.30 177.61 # ± 0.92 Protein (mg/DCW) 221.36 ± 18.35 262.02 ± 35.89 220.99 ± 18.58 230.70 ± 33.02 253.03 ± 29.00 Total fatty acid (mg/DCW) 34.68 ± 0.34 42.19 ± 1.92 46.85 ± 0.83 37.87 # ± 1.16 34.90 ## ± 1.84 The values shown are the averages of three biological replicates and three measurement replicates. *Difference between transformants and wild types,*p < 0.05,** p < 0.01. #Difference between the experimental group and the control group with addition of 4-HB,#p < 0.05,##p < 0.01. The level of intracellular neutral lipids in transformant significantly increased by 58.18% ( p < 0.05) ( Fig. 4 a). We found each fatty acid composition increased in transformant ( Table 2 ). The total fatty acid content in transformant was 42.18 mg/g DCW, which significantly increased by 10.56% compared with the wild type ( p < 0.01) ( Fig. 4 b). In addition, the protein and glycogen content of transformant was increased by 8.16% and 18.37% respectively ( Fig. 4 c and d). Based on the above results, we can speculate that the lepgt overexpression could transfer more carbon sources to the synthesis pathway of lipid and protein in Synechocystis . Fig. 4 Intracellular metabolite contents of Synechocystis PCC 6803. (a): the fluorescence intensity of the neutral lipid; (b): the production of total fatty acid; (c): the production of glycogen; (d): the production of protein. The group of 0 mM was wild type Synechocystis which 1% ethanol was added. Standard errors was calculated from three biological independent experiments; asterisks represent significant differences (* p < 0.05, ** p < 0.01). Fig. 4 Table 2 Fatty acid profiles of Synechocystis PCC 6803 with different treatments. Table 2 Fatty acid type WT LePGT Control 1 mM 4-HB 2 mM 4-HB (mg/g DCW) C16:0 23.40 ± 2.38 25.81 ± 1.18 32.78 ± 1.74 27.32 # ± 3.31 22.27 # ± 2.71 C16:1 1.00 ± 0.2 1.01 ± 0.118 1.54 ± 0.39 2.16 ± 0.33 3.21 # ± 0.32 C18:0 1.46 ± 0.26 1.76 ± 0.343 1.14 ± 0.62 0.43 ± 0.077 0.55 ± 0.13 C18:1 3.47 ± 0.868 4.13 ± 0.487 3.26 ± 1.79 0.42 ## ± 0.052 0.88 ## ± 0.12 C18:2 3.96 ± 0.521 4.14 ± 0.472 3.35 ± 0.75 4.29 # ± 0.44 3.33 ± 0.54 C18:3 4.90 ± 0.90 5.33 ± 0.319 6.23 ± 0.30 5.90 ± 0.60 4.66 ± 0.81 The values shown are the averages of three biological replicates and three measurement replicates. # Difference between the experimental group and the control group with addition of 4-HB, # p < 0.05, ## p < 0.01. 3.6.2 The effect of different increments of PQ on intracellular metabolites 4-HB addition dramatically increased the neutral lipid content by 54.82% at 1 mM and by 41.35% at 2 mM ( Fig. 4 a). As shown in Fig. 4 b and Table 2 , the intracellular fatty acid content of Synechocystis was decreased by the addition of exogenous 4-HB. Compared to the control group, the total fatty acids content was decreased by 20.34% with 1 mM 4-HB addition and decreased by 26.59% with 2 mM 4-HB addition. Significant decreases of C16:0 and C18:1 were observed with 1 mM 4-HB addition. The yield of C16:1 and C18:2 enhanced with 1 mM 4-HB addition. In addition, 4-HB dropped the levels of C18:0 and C18:0 at 1 mM. The content of C16:1 was significantly increased, but the content of other fatty acid decreased after a 2 mM 4-HB treatment ( Table 2 ). Glycogen content was increased in Synechocystis with 4-HB addition (1 mM, 34.92%; 2 mM 16.78%) ( Fig. 4 c). Compared with the control group, the level of protein was slightly decreased by 5.98% at 1 mM, but increased by 15.80% at 2 mM ( Fig. 4 d). According to the results, we can infer that the addition of 4-HB could regulate carbon flow, and its impact on carbon flow varies with concentration ( Fig. 5 ). Fig. 5 Scheme illustrating metabolic regulation of Synechocystis PCC 6803 upon photosynthetic and respiratory electron transport chain mediated by plastoquinone. Red: Respiratory electron transport chain; Green: Photosynthetic electron transport chain; Thick line means the pathway is enhanced. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 5 Due to the different physiological metabolic pathways, the phenotype changes of microalgae caused by the addition of compounds are different. For example, when Chlamydomonas reinhardtii was treated with exogenous low-concentration 4-HB, the growth was significantly stimulated and the lipid peroxidation significantly inhibited [ 50 ]. Low-concentration 4-HB treatment can promote the growth of Chlorella and the synthesis of intracellular nucleic acids, cytochromes and soluble proteins [ 51 ]. In this work, it was worth noting that the addition of 1% ethanol could significantly promote the accumulation of neutral lipid and fatty acid, as ethanol may affect the stability and fluidity of cell membrane of Synechocystis [ 52 ]. In addition, 4-HB had little effect on growth, intracellular protein and cytochrome content in Synechocystis at concentrations of both 1 mM and 2 mM. The addition of a range of chemicals, such as 4-HB, salicylic acid, traumatic acid, can promote cell growth and metabolism in green algae ( Chlamydomonas reinhardtii , Chlorella spp.). While adding higher concentrations of such compounds could have a significant inhibitory effect on cell growth and intracellular anabolic pathways. There is not a linear relationship between the changes of microalgae phenotype and the concentration of the compounds [ 32 , 51 ]. The phenotypic changes, such as intracellular fatty acid content, do not show a linear change in Synechocystis treated with different concentration of exogenous ethylene [ 53 ]. In this study, 4-HB enhanced the contents of intracellular glycogen and the neutral lipid at 1 mM which was higher than those at 2 mM 4-HB. In summary, the overexpression of lepgt in Synechocystis could competitively drain 4-HB and GPP, significantly reduce the content of PQ and promote the synthesis of intracellular fatty acids, neutral lipids, and glycogen. By the addition of exogenous 4-HB, we found that different increment of intracellular PQ could regulate the metabolism of Synechocystis, showing different contents of intracellular fatty acid, glycogen, and neutral lipid. The results showed that the small changes of PQ content in Synechocystis can achieve precise regulation of physiology and metabolism. The further experiment can be combined with metabolomics to further explore how the changes of PQ content cause different effects on intracellular metabolic pathways. The strategies of genetic engineering and compound addition in this work provide a new strategy for promoting the synthesis of active substances in Cyanobacteria."
} | 8,436 |
30404317 | PMC6189925 | pmc | 6,250 | {
"abstract": "This article describes a new way to explore neuromorphic engineering, the biomimetic artificial neuron using microfluidic techniques. This new device could replace silicon neurons and solve the issues of biocompatibility and power consumption. The biological neuron transmits electrical signals based on ion flow through their plasma membrane. Action potentials are propagated along axons and represent the fundamental electrical signals by which information are transmitted from one place to another in the nervous system. Based on this physiological behavior, we propose a microfluidic structure composed of chambers representing the intra and extracellular environments, connected by channels actuated by Quake valves. These channels are equipped with selective ion permeable membranes to mimic the exchange of chemical species found in the biological neuron. A thick polydimethylsiloxane (PDMS) membrane is used to create the Quake valve membrane. Integrated electrodes are used to measure the potential difference between the intracellular and extracellular environments: the membrane potential.",
"introduction": "1. Introduction Millions of people worldwide are affected by neurological disorders which disrupt connections between brain and body, causing paralysis or affecting cognitive capabilities. The number is likely to increase over the next few years and current assistive technology is still limited. In recent decades, extensive research has been devoted to brain-machine interfaces (BMIs), and neuroprostheses in general [ 1 , 2 , 3 ], working towards effective treatment for these disabilities. The development of these devices has had and, hopefully, will continue to have a profound social impact on these patients' quality of life. These prostheses are designed on the basis of our knowledge of interactions with neuronal cell assemblies, taking into account the intrinsic spontaneous activity of neuronal networks and understanding how to stimulate them into a desired state or produce a specific behavior. The long-term goal of replacing damaged neural networks with artificial devices also requires the development of neural network models matching the recorded electrophysiological patterns and capable of producing the correct stimulation patterns to restore the desired function. The hardware setup used to interface the biological component is a biomimetic neural network system implementing biologically realistic neural network models, ranging from the electrophysiological properties of a single neuron to large-scale neural networks. This research field is named “neuromorphic engineering”. The neuromorphic engineering is a new emerging interdisciplinary field merging biology, physics, mathematics, computer sciences, and engineering approaches to design biomimetic artificial neural systems. These artificial neural networks emulate the electrical activity of biological neural networks and their goal is to mimic and/or replace the organic ones. Most of these systems are silicon-based [ 4 , 5 ]. The main goals of those systems are the design of tools for biomedical applications, like neuroprostheses [ 6 ], and the understanding of the human nervous system [ 7 , 8 ]. The use of silicon neurons brings some bio-compatibility issues (rejection, power consumption). To circumvent those issues, a new way of neuromorphic engineering should be explored: the artificial neural systems based on microfluidic techniques ( Figure 1 ). The aim is to design a neuromimetic architecture of one neuron based on the use of microfluidic techniques [ 9 ] and microsensor integration. In Figure 1 , the microfluidic neuron is between the silicon neuron and the biological one. It could offer a good trade-off with more biological plausibility than the silicon neuron, and to make hybrid experiments in an easier manner (bi-directional communication between a living neuron and an artificial one). To our knowledge, this new methodology does not exist yet in the state of the art. In this article, we will first describe the novelty and the state of the art about biomimetic artificial neurons, then discuss microfluidic neuron modelling and its design; finally, we will present the first results and perspectives of this work.",
"discussion": "5. Discussion 5.1. Hybrid Experiments In a final step, hybrid experiments in the same chip were designed: neurons culture in one part of the PDMS device and artificial neurons in the other part. This PDMS device is described in Figure 16 . Electrodes on glass are used for stimulation and recording of biological neurons. The microfluidic neuron is used for stimulating the biological part by the activation or inactivation of the Quake valves. This device is ready for the open-loop (microfluidic neuron to biological neuron) experiments. For the closed-loop (bidirectional communication) experiment, an electrical board for recording and detecting the biological spiking activity is needed. 5.2. New Design Ideas A key point of the design of such a microfluidic neuron is the membrane exchange between the intracellular and extracellular environments. A membrane made of Nafion provides a selectivity with respect to the charge of the ions in passing only cations (K + and Na + in our case). Selectivity for specific ions is not possible with this membrane. The integration of lipid bi-layers [ 36 ] would alleviate this problem, and constitute an important method to investigate. The miniaturization of the neuron to approach the biological dimensions (of the order of tens of microns), represents an additional step to the technological constraints, particularly on the integration of the selective membrane. The networking of microfluidic neurons should begin before the phase of miniaturization. This will adapt to any architectural changes required to obtain neural networks with dimensions similar to those of biological neurons. A matrix of microfluidic neurons can be designed in the near future. The plasticity of material integration is currently under investigation in order to add synapses between the different artificial neurons. A better miniaturization of the Quake valve can be accomplished. Bursting behavior generated by the microfluidic neuron will be more accurate and with higher frequency. The use of a piezoelectric valve could be a direction to explore. 5.3. Microfluidic Neuron as a Stimulator Another potential approach will be to use the microfluidic neuron as a stimulator for biological neurons. Given that the action potential of the microfluidic neuron has the same characteristics than biological ones, and the ionic concentrations in the microfluidic neuron and in the neuron culture medium are similar, we foresee that the extracellular chamber of the microfluidic neuron and the neuron culture system can be merged. The microfluidic neuron as a stimulator is, in our opinion, one of the most promising methods to explore in neuromorphic engineering."
} | 1,730 |
20300174 | PMC2836372 | pmc | 6,253 | {
"abstract": "The success of social animals (including ourselves) can be attributed to efficiencies that arise from a division of labour. Many animal societies have a communal nest which certain individuals must leave to perform external tasks, for example foraging or patrolling. Staying at home to care for young or leaving to find food is one of the most fundamental divisions of labour. It is also often a choice between safety and danger. Here we explore the regulation of departures from ant nests. We consider the extreme situation in which no one returns and show experimentally that exiting decisions seem to be governed by fluctuating record signals and ant-ant interactions. A record signal is a new ‘high water mark’ in the history of a system. An ant exiting the nest only when the record signal reaches a level it has never perceived before could be a very effective mechanism to postpone, until the last possible moment, a potentially fatal decision. We also show that record dynamics may be involved in first exits by individually tagged ants even when their nest mates are allowed to re-enter the nest. So record dynamics may play a role in allocating individuals to tasks, both in emergencies and in everyday life. The dynamics of several complex but purely physical systems are also based on record signals but this is the first time they have been experimentally shown in a biological system.",
"introduction": "Introduction Ant societies are shaped by selection that operates, in part, at the level of the colony [1] , so the success of the individual is intimately bound to that of its colony. Outside-nest work is dangerous and the rate of attrition of outside-nest workers through predation or adverse environmental conditions is often high. The life-cycles of ant societies are dominated by growth or decline [2] . Thus they are rarely at a steady state and are typically non-stationary. Here we induce non-stationarity by permanently eliminating all ants that exit the nest and compare these colonies with controls in which ants can freely leave and re-enter the nest. We use analytical methods developed for the analysis of out-of-equilibrium physical systems to explore the nature of the mechanism governing the decisions of individual ants to leave the nest. Indeed, biological systems are, like other systems in Nature, generically non-equilibrium systems since they are not isolated from external influences and continuously have a flux of mass or energy passing through them [3] . The null model for a system in which successive events are drawn from a diminishing pool is one with an exponentially declining event rate, as in radioactive decay. In this scenario there are either no interactions between the components or interactions between the components are not correlated with decay events. The simplest form of radioactive decay is one in which all the components have an identical decay probability, which can be modelled as a homogeneous Poisson process. Obviously ants are not all identical, so for our null model we implement a heterogeneous Poisson process by assuming that the component parts (the ants) vary in their decay (i.e. exit) probabilities. An alternative scenario that produces rapidly decreasing event rates is one in which events are triggered when a fluctuating variable - the record signal - exceeds its historical ‘high water mark’. If the record signal fluctuates randomly, the increment between successive record values becomes progressively smaller and the rate at which new records accrue drops off according to the inverse of time [4] , [5] . Hence the rate of change is a function of the age of the system. An intuitive example of a rapidly decelerating record time-series (albeit one that is probably not based on record signals as defined in complex systems) is the accumulation of human sporting records, where the rate at which new records accumulate depends largely on the age of the sport [6] , [7] . All cases in which fluctuating record signals trigger events, include strong interactions between the component parts and involve long-range correlations that span the entire system [8] , [9] , [10] , [11] . While exponential decay is characterised by Poisson statistics in linear time, record dynamics is characterised by Poisson statistics in logarithmic time [4] , [5] , [8] , [12] , [13] , [14] . What mechanism can generate such log-Poisson statistics? A fluctuating record signal will only produce log-Poisson statistics if each successive value of the underlying fluctuating signal is independent of its predecessors. Independence of the fluctuating record signal leads the record times to be uncorrelated in logarithmic time, so the record value at time log (T k ) is independent of previous records at time log (T k-n ). Crucially, the distribution of the underlying fluctuating signal from which the record signal is derived, must not change over time. Quite remarkably, irrespective of the probability distribution of the underlying fluctuating signal, records will accrue at a logarithmically decreasing rate [5] . We test whether nest leaving activity is compatible with either of two models of rapidly decelerating events: exponential decay as a null model or record dynamics. We further test the effects of heterogeneous units and varying colony size on the exponential decay model through a simulation parameterised from data.",
"discussion": "Discussion We found that ant exits were compatible with a record dynamics process while the null model could not reproduce the observed statistics. Therefore, the temporal pattern of ant exits implies that ant-ant interactions and fluctuating record signals govern individual nest-leaving decisions. Log-Poisson statistics are only found when the system is in a statistically non-stationary or ‘transient’ state [4] , [5] . Thus the demonstration of record dynamics implies that the colony is not in a steady state, i.e. not at equilibrium, even when the population size is stationary. Such a mechanism might be adaptive for a dangerous task such as leaving the nest, because individuals may only do so when the demand reaches an unprecedented level, for example, the brood may be hungrier and the adult workforce thirstier than ever before. Intriguingly, record dynamics were present both when ants were removed and in the non-removal control. In the treatment the colony population size was forced to decrease. Similarly, in the control the definition of an event as the exit of any ant previously not seen leaving the nest effectively creates a diminishing sub-population of individuals that have yet to be observed leaving the nest. This sub-population is the equivalent of the population remaining in the nest in the experiment where ants are prevented from returning, as it also undergoes a decline towards zero, when all ants in the colony would have been observed leaving the nest at least once. As the same qualitative pattern was found under both the removal and non-removal condition, interactions with ants that have not recently returned to the nest seem sufficient for the generation of log-Poisson statistics. So fluctuating record signals and ant-ant interactions may regulate individual exit decisions irrespective of whether external workers return to the nest. If the temporal pattern of exits appears to be qualitatively identical under both benign and hostile environmental conditions, are there any quantitative differences? When workers are prevented from returning, the distribution of τ is significantly more skewed towards larger values ( Fig. 8 ). In honeybees [18] , bumblebees [19] , and other ants [2] , stimuli derived from interactions with returning individuals up-regulate the exit rate. However, to our knowledge this is the first evidence that the complete severance of this feedback loop results in down-regulation of the exit rate. It should be emphasised that the statistical mechanism described in the high water-mark record-dynamics model, is a macroscopic summary of the processes occurring at the microscopic level. It does not describe the specific microscopic processes. However scenarios in which fluctuating record signals trigger events are always associated with long-range correlations that span the system. These correlations arise out of short-range interactions between the components [5] , [10] . Since the designation of a new record depends on the preceding sequence of record values, and because through local interactions, the components share this collective history, all components involved in a process controlled by record dynamics must necessarily be strongly correlated [8] , [9] , [10] , [11] . Intermittent dynamics associated with event rates that decelerate rapidly, but non-exponentially, have been described in 10000 generation experiments of bacterial evolution [20] , declining extinction rates [9] , [21] , the ‘Tangled Nature’ model of macroevolution [4] , [10] , fluctuating commodity prices [22] , type-II superconductors [11] , colloidal gels [14] , and spin glasses [8] . To our knowledge this is the first experimental evidence for record dynamics in living systems, that is, those that have been shaped by Darwinian evolution. The concept of complex systems was developed in the physical sciences to explain the emergence of macroscopic phenomena from the interactions of large numbers of microscopic components. In biology this approach has greatly aided our understanding of collective phenomena such as decentralised control and pattern formation [23] . The theory of self-organisation was originally developed to explain pattern formation in stationary physical systems - those at a statistical steady state. However the physical systems that decelerate according to record dynamics are manifestly non-stationary. The identification of the record signal even in purely physical systems is a hard problem, indeed the only case in which the record signal has been unequivocally identified (i.e. thermal energy) is the Edwards-Anderson spin-glass [4] . We have described a biological social system that displays the same statistics as found in many non-stationary physical systems governed by decelerating record dynamics, and hence infer that a similar statistical mechanism is in operation in the ants. What could be the individual mechanism underlying the collective record dynamics? One plausible scenario is that individual variation in the perception of the record signal can lead to variation in the memory of the standing record amongst individuals. We have demonstrated that decision-making in ant societies may originate from the combination of fluctuating record signals and ant-ant interactions. This suggests that further understanding of signals and interactions between individuals within the colony could elucidate not only the organisation of insect societies but also facilitate the understanding of general principles of system organisation. The challenge for biologists is to identify these signals and interactions by quantifying the behaviour of the individual colony members over time. This opens a future avenue for new manipulative experimentation and theory."
} | 2,797 |
37843294 | PMC10746200 | pmc | 6,254 | {
"abstract": "ABSTRACT In its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa . However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL , coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system. IMPORTANCE Single-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa .",
"introduction": "INTRODUCTION Many bacterial species utilize quorum sensing (QS) to coordinate social activities in response to changes in population density. QS is a cell-to-cell chemical communication process based on the extracellular release and perception of signal molecules. QS signal molecules are produced at basal levels at low cell density and accumulate in the medium as the bacterial population grows, until reaching a threshold concentration. The quorum is defined as the cell population density turning point at which signal molecules bind to and activate cognate transcriptional regulators or sensor kinases, leading to coordinated genetic reprogramming in all the cells of the population. A common feature of these systems is the positive effect exerted by the activated QS receptor on the production of the QS signal molecule, which generates a positive feedback loop that accelerates QS activation ( 1 – 3 ). As one of the main systems involved in the control of bacterial collective behaviors, QS has long been regarded as a process homogeneously activated by the entire population in a highly synchronized way. However, gene expression analyses at the single-cell level suggest that this assumption is not the rule. Indeed, recent evidence indicates that QS activation is not always synchronous in single cells of a bacterial population and that the expression level of QS genes could considerably differ from one cell to another, sometimes resulting in a bifurcation of the population in quorate and non-quorate subpopulations ( 4 – 14 ). In some cases, heterogeneity has been shown to be transient and limited to the early phases of QS activation, with bacterial population subsequently converging to a homogeneous quorate state ( 11 , 12 ). In other cases, inter-individual QS heterogeneity has been reported to persist also at high cell densities ( 4 , 10 , 15 ). The investigation of cell-to-cell variability of QS activation is more complicated in bacteria possessing multiple and interconnected QS systems. As an example, Vibrio harveyi produces three different signal molecules, and the output QS response was found to be homogeneous only when all signals were produced at high levels, while heterogeneity arose in the absence of any one of the three signal molecules ( 16 ). A paradigmatic case of bacterial species possessing multiple QS systems is the opportunistic human pathogen Pseudomonas aeruginosa , in which three interconnected QS circuits, known as las , rhl , and pqs , are employed to finely modulate the expression of hundreds of genes in response to cell density and other environmental and metabolic cues. Notably, genes coding for several virulence factors and involved in biofilm formation is under the control of one or multiple QS systems in P. aeruginosa , highlighting QS synthase and receptor proteins as promising targets for the development of antivirulence drugs reducing P. aeruginosa pathogenicity ( 17 , 18 ). Heterogeneous QS activation has recently been addressed in P. aeruginosa ( 14 , 19 ). Rattray and co-workers observed that activation of the QS-controlled gene lasB shows a heterogeneous and graded response to variations in the population density, indicating that there is no critical cell concentration triggering QS-dependent response ( 19 ). Moreover, their data indicate that the population diverges into subpopulations of quorate and non-quorate cells that also coexist at high cell density ( 19 ). Conversely, Jayakumar and colleagues found transient segregation of cells into discrete subgroups with distinct QS-related gene expression states at low cell density, with all the cells of the population converging to the quorate state at high cell density ( 14 ). Overall, despite no consensus being reached on the occurrence of non-quorate cells at high cell density, these seminal studies demonstrate that cell-to-cell variation in the QS activation state during P. aeruginosa growth significantly exceeds intrinsic gene expression noise ( 20 , 21 ). At present, this remarkable finding lacks a solid molecular explanation. Positive feedback loops, as those integrated into QS circuits, can generate or amplify gene expression heterogeneity ( 22 – 24 ), and also, negative feedback loops can favor cell-to-cell variation in gene expression ( 22 , 25 , 26 ). Youk and Lim showed via single-cell level analysis of a budding yeast strain engineered with a synthetic QS circuit that heterogeneity in the activation of this synthetic signaling system arose by integrating a positive feedback loop, which tends to amplify signal molecule production, with a negative regulatory system, which limits signal molecule availability ( 27 ). This regulatory architecture resembles the P. aeruginosa las QS system, in which the positive feedback loop generated by the LasR transcriptional regulator in complex with the N -(3-oxododecanoyl)-L-homoserine lactone (3OC 12 -HSL) signal molecule is counteracted by the negative feedback loop generated by RsaL ( Fig. 1 ). Indeed, in the las system, the 3OC 12 -HSL signal molecule produced by the LasI synthase binds to and activates the intracellular receptor LasR, resulting in the regulation of diverse target genes ( 28 – 30 ). When interacting with the rsaL-lasI bidirectional promoter, the LasR/3OC 12 -HSL complex increases lasI transcription, thus generating the canonical positive feedback loop that enhances 3OC 12 -HSL production but also triggers transcription of the rsaL gene. RsaL, in turn, represses lasI transcription and its own expression when it binds to the rsaL-lasI bidirectional promoter on a palindromic sequence centered between the lux -box for LasR binding and the lasI transcriptional start site ( Fig. 1 ) ( 31 – 34 ). Hence, on one hand, the LasR/3OC 12 -HSL complex promotes 3OC 12 -HSL synthesis, thus boosting its activation state and the QS response, while on the other, it limits 3OC 12 -HSL production and QS activation by stimulating the transcription of the QS negative regulator gene rsaL . Although apparently contradictory, such a regulatory mechanism, known as incoherent feed-forward loop, is widespread among biological systems ( 35 ). It is known to confer robustness to lasI gene expression with respect to fluctuations in the levels of LasR ( 36 ). Overall, this evidence prompted us to investigate the impact of RsaL on QS heterogeneity in P. aeruginosa . Fig 1 The P. aeruginosa rsaL-lasI gene locus . Schematic representation of the DNA region encompassing the rsaL and lasI genes in P. aeruginosa ( 37 ). Solid green arrow indicates activation (+); red T-line indicates negative regulation (−); the red and green boxes in the rsaL-lasI intergenic region indicate RsaL and LasR binding sites, respectively ( 32 , 33 ). On the bottom, the rsaL-lasI bidirectional promoter region and the corresponding P lasI * variant (RsaL-binding negative) are depicted: the −35 and −10 regions are indicated; the LasR-binding site is green shadowed; the RsaL-binding site is red shadowed; transcriptional start site is underlined; repeats forming the palindromic sequence bound by RsaL are indicated by arrows; nucleotides whose substitution abrogates RsaL binding to the rsaL-lasI intergenic region are in bold; triangles indicate nucleotide substitutions introduced in the P lasI * variant. By using single-cell level gene expression analyses, we show that the RsaL-driven negative regulation of lasI transcription increased cell-to-cell variation in the activation state of the las QS system and caused bifurcation of the population into subsets of quorate and non-quorate cells that also coexist at high cell density. Interestingly, the same phenotypes were not displayed by the rhl QS system that is not controlled by an incoherent feed-forward loop.",
"discussion": "DISCUSSION Over the years, single-cell level studies revealed that heterogeneity in the activation of QS systems is not an unusual feature ( 48 – 50 ). However, the molecular basis of such heterogeneity has seldom been investigated. Here, we identify RsaL as a key molecular actor determining cell-to-cell variation of lasI expression in P. aeruginosa and demonstrate that binding of this negative regulator to the rsaL-lasI bidirectional promoter is pivotal for heterogeneous activation of the las QS system. In line with findings of Rattray and collaborators, which monitored the QS-regulated P lasB promoter at the single-cell level under conditions of varying carbon availability ( 19 ), we found that las system activation was graded with respect to increasing levels of cell densities. Such recent evidence about graded QS induction in P. aeruginosa , obtained via single-cell level analysis, should lead to reconsidering the canonical vision of the threshold-dependent QS activation, as in some cases, QS might not necessarily be characterized by an “OFF/ON” status determined by a specific level of cell density. We also found that the PAO1 population tends to be less heterogeneous with respect to P lasI activation proceeding with growth, in accordance with reference 14 . However, a certain level of heterogeneity also persisted into the stationary phase, where a significant fraction of cells did not activate the P lasI promoter. The elegant analyses performed by Rattray and collaborators in minimal M9 medium supplemented with MOPS showed a similar pattern of activation of the lasB promoter for the higher cell densities they considered ( 19 ), in line with our findings showing the coexistence of quorate and non-quorate cells in stationary phase populations. Since the maximum OD 600 reached by P. aeruginosa populations used by Rattray and colleagues was <0.8 ( 19 ), it may not have been sufficient to fully trigger QS. Here, we conducted our analyses in the rich medium LB-MOPS, in which PAO1 cultures reached an OD 600 of ca. 4 in the stationary phase, and at such high cell densities, we still observed the coexistence of quorate and non-quorate cells. The fact that not all the cells of a population enter the quorate state at high cell densities is not limited to P. aeruginosa . Indeed, this phenomenon has also been described for Pseudomonas syringae pv. syringae , Xanthomonas campestris , V. harveyi , and Sinorhizobium fredii ( 4 , 9 , 10 ). In this study, neither the quorate cell fraction nor the degree of heterogeneity was particularly affected when saturating levels of synthetic 3OC 12 -HSL were added to P. aeruginosa cultures. Also, P. syringae and X. campestris have been demonstrated to retain QS heterogeneity following exogenous signal molecule addition ( 10 ). On the other hand, in S. fredii ( 9 ) and V. harveyi ( 4 ), QS synchronization occurred in response to external signal molecule provision, leading the authors to hypothesize that, in these bacteria, QS heterogeneity could be ascribed to an unsaturated state of signal molecule receptor. In P. aeruginosa , saturating 3OC 12 -HSL levels do not shift the population to a homogeneously quorate state, meaning that unsaturated LasR levels cannot account for heterogeneity in QS activation. Moreover, we previously showed that the incoherent feed-forward loop integrated into the las system confers robustness with respect to fluctuations in LasR levels to promoters controlled by both LasR/3OC 12 -HSL and RsaL ( 36 ). Hence, it is reasonable to hypothesize that the incoherent feed-forward loop generated by LasR and RsaL could confer robustness to P lasI activity also with respect to fluctuations in 3OC 12 -HSL levels. Furthermore, the inability of exogenous signal molecule provided at saturating levels to induce QS activation at the onset of bacterial growth could rely on the action of the QscR, QslA, and/or QteE anti-activators, which keep the LasR regulator in an inactive state not competent for 3OC 12 -HSL binding at low cell density ( 51 – 54 ). The transcriptional regulator MvaT could also play a role in this phenomenon, as exogenous 3OC 12 -HSL can significantly advance the expression of las -controlled genes in a mvaT mutant but not in wild-type PAO1 ( 55 ). Our work describes for the first time the involvement of RsaL in controlling the transition of non-quorate cells to the quorate state. Likewise, the LuxO repressor, through destabilization of the luxR transcript, was shown to increase heterogeneity in bioluminescence emission in V. harveyi ( 4 ). Transient co-existence of quorate and non-quorate cells in the ∆ rsaL mutant during the exponential phase of growth could possibly be attributed to intrinsic transcriptional noise. However, the possibility that factors other than RsaL could contribute to cell-to-cell variation in the activation state of the las QS system at low cell density should be considered. Interestingly, evidence for heterogeneous lasR gene expression has been provided during exponential growth ( 14 ), indicating that the positive feedback loop generated by the LasR/3OC 12 -HSL complex may contribute to las system heterogeneity at the onset of QS activation. In this context, it is worth mentioning that a positive feedback loop has been shown to increase heterogeneity of QS activation in Bacillus subtilis ( 24 ). However, a study from Scholz and Greenberg reported that the las QS response shows greater synchrony in wild-type P. aeruginosa than in an engineered strain lacking the positive feedback loop controlling 3OC 12 -HSL synthesis, in which the signal molecule was constantly produced regardless of the population cell density ( 40 ). It is relevant to note that population-based and single-cell level analyses could lead to different interpretations of the same phenomenon. According to the previously proposed model of las system activation and QS homeostasis ( 34 ), the LasR/3OC 12 -HSL complex can drive transcription of the rsaL and lasI genes at the quorate cell density. The LasR/3OC 12 -HSL-dependent positive feedback loop increases both 3OC 12 -HSL synthesis and RsaL levels, to the point that RsaL interacts with its DNA-binding site on the rsaL-lasI bidirectional promoters. This abrogates transcription of both lasI and rsaL , thus limiting 3OC 12 -HSL production to productive levels at high cell density. Although this was the conclusion that clearly emerged when las QS activation was analyzed at the population level, the single-cell level analyses performed here are consistent with an alternative mechanistic model. When the LasR/3OC 12 -HSL complex binds to the rsaL-lasI bidirectional promoter, stochastic events could result in asymmetric transcription of either lasI or rsaL in individual cells instead of homogeneous expression of these genes in the whole population. The negative regulation exerted by RsaL on lasI transcription could reinforce the variance between RsaL and LasI production among cells, further limiting QS activation in the proportion of cells characterized by high rsaL expression levels. In this context, the uneven LasR/3OC 12 -HSL-dependent transcription of lasI and rsaL appears to be a primary driver of QS heterogeneity. The absence of correlation between lasI and rsaL expression in single P. aeruginosa cells supports this model. Activation of the rhl system at the single-cell level follows a different trend compared to the las system, as this QS system appeared to be homogeneously activated by all cells starting from the exponential growth phase and was unaffected by RsaL. This finding was unanticipated, as it is generally accepted that the las system exerts positive transcriptional control over both rhlR and rhlI ( 46 ). However, it must be considered that previous studies showed deregulation of the las QS system but not of the rhlR and rhlI transcripts in a PAO1 strains with an rsaL deletion or overproducing RsaL ( 34 , 47 ) and that the reciprocal control of the las and rhl QS systems appears to be more complex than predicted in previous studies ( 56 – 59 ). Overall, data showing low heterogeneity in the activation state of the rhl system, which is not controlled by an incoherent feed-forward loop, reinforce the importance of the RsaL-dependent negative regulation in determining cell-to-cell variation in the las QS activation state. Since RsaL homologs are present in the QS systems of several bacterial species ( 60 ), it will be interesting to employ single-cell level analyses to investigate the impact of RsaL on QS activation in bacteria other than P. aeruginosa and to clarify if heterogeneity is a common property of QS systems characterized by non-RsaL negative regulators, such as TraM, RsaM, AbaM, and PqsE ( 61 – 65 ). At present, the significance in P. aeruginosa physiology and ecology of maintaining non-quorate cells at high cell density remains unclear. It is known that coexistence of different phenotypic variants in a monoclonal bacterial population could favor division of labor between individuals ( 66 , 67 ). In other cases, phenotypic heterogeneity could represent a bet-hedging strategy, as part of the population could exhibit a different “pre-adapted” phenotype that will ensure the survival or a better adaptation of part of the bacterial population in case of sudden environmental changes ( 67 , 68 ). In general, P. aeruginosa is an extremely versatile microorganism that integrates a number of environmental signals through multiple regulatory networks for optimally adapting to different niches ( 69 , 70 ). Among the diverse controlled phenotypes, QS can mediate P. aeruginosa transition to different lifestyles (as an example, from the planktonic to the biofilm state, and vice versa), and such transitions could lead individual cells to face consistent and rapid changes in the surrounding environment. It should also be considered that the synthesis of QS signal molecules and QS-regulated exoproducts imposes a metabolic burden on P. aeruginosa ( 71 , 72 ) and that QS activation provides a fitness benefit to this bacterium mainly at high cell density ( 73 ). In this regard, maintaining a fraction of cells in a non-quorate state at high cell density could be beneficial if sudden changes in environmental conditions occur, including dilution of the bacterial population. Indeed, non-quorate cells could be better adapted than quorate cells at low cell density, as they could avoid a lag in the shut-down of 3OC 12 -HSL production if suddenly diluted, hence limiting short circuiting of the las QS system, i.e., intracellular self-activation of LasR ( 41 ). It should be also considered that, even if non-quorate cells are still able to respond to QS signal molecules, high levels of RsaL in non-quorate cells could limit their metabolic burden, as RsaL directly represses the expression of several LasR-dependent genes other than lasI ( 34 , 36 , 44 , 47 ). Notably, despite frequent isolation of P. aeruginosa clinical and environmental isolates with lasR inactivating mutations and/or rewiring of the QS hierarchy ( 58 , 74 – 76 ), the rsaL gene sequence is conserved among P. aeruginosa strains ( 77 ), suggesting that the negative regulatory loop generated by RsaL may be important for P. aeruginosa fitness in both clinical and natural settings."
} | 5,477 |
36777616 | PMC9909818 | pmc | 6,256 | {
"abstract": "It is challenging to convert the superhydrophobic surfaces\nof iron-based\namorphous films into hydrophilic surfaces through surface treatment.\nIn this study, a novel, environmentally friendly method is used to\nchange the superhydrophobic surfaces of Fe 78 Si 13 B 9 amorphous alloy films, which include their rougher\nand smoother surfaces. The boron element in the films reacted with\nthe flavonoids and anthocyanins in the solution to create organic\nconversion membranes and organic boronizing naphthoquinone derivatives\non the surfaces of the films when they were dipped in tea polyphenol\naqueous solution at 80 °C for 60 min. On the rougher surface\nand the smoother surface, the organic conversion membranes had thicknesses\nof about 10 and 3 μm, respectively. When iron-based amorphous\nalloy films were employed as soft magnetic materials to create electronic\nand electrical devices, the packaging issue caused by low wettability\nwith epoxy resin had been resolved because both the side surfaces\nof modified films had good wettability with epoxy resin. In addition,\nthe magnetic surface effect of modified films was significant. After\nsurface treatment, the inductance value of the film decreased by more\nthan 25%. The magnetic surface effect of iron-based amorphous films\ncan be applied to the preparation of tea sensors, and the sensor can\nachieve the “one to one” high precision test of “one\ntea curve”. The magnetic surface effect of the film provides\na quick, simple, lower cost, and strong anti-interference idea for\nthe rapid detection of tea polyphenols.",
"conclusion": "5 Summary and Conclusions An environmentally\nfriendly surface treatment technology, tea polyphenol\ntreatment technology, is adopted to modify the superhydrophobic surfaces\nconsisting of the smoother surface and the rougher surface of Fe 78 Si 13 B 9 amorphous alloy films. Organic\nboronizing naphthoquinone derivatives with a uniform thickness were\nformed on the surfaces of films. The thickness of organic conversion\nmembranes was approximately 10 μm on the rougher surface and\n3 μm on the smoother surface. Both the side surfaces of\nmodified films have good wettability\nwith epoxy resin, which solves the packaging problem when Fe 78 Si 13 B 9 amorphous alloy films are adopted for\nelectronic and electrical devices as a soft magnetic material. After the surface cation, the modified films of Fe 78 Si 13 B 9 amorphous alloy have a significant magnetic\nsurface effect. The magnetic surface effect increases with improving\nthe surface treatment temperature. At the surface treatment temperature\nof 80 °C, the film inductance decreased by more than 25% when\nthe tea polyphenol concentration was ≥230 mg/g. The magnetic\nsurface effect of iron-based amorphous films can be applied to the\npreparation of tea sensors. The sensor can achieve the “one\nto one” high precision test of “one tea curve”,\nproviding a fast, simple, low-cost, and strong anti-interference scheme\nfor the detection of tea polyphenol content and evaluation of tea\nquality.",
"introduction": "1 Introduction Because of their unique\nmicrostructure, excellent mechanical properties,\nand soft magnetic properties and having the advantages of high magnetic\nconductivity, high saturation magnetic sensitivity, low coercivity,\nlow iron-loss value, and good thermal stability and corrosion resistance,\niron-based amorphous alloy films are widely used as soft magnetic\nmaterials in the manufacture of transformers, 1 sensors, 2 , 3 and other devices. 4 − 6 When iron-based\namorphous alloy films are used as soft magnetic materials to make\nelectronic and electrical devices, they need to be encapsulated with\norganic resin (such as epoxy resin). Therefore, it is very important\nto modify the surface of the films to improve their wettability to\nresin. 7 , 8 On the other hand, using the sensitive\ncharacteristics of the amorphous\nstructure combined with the characteristics of film toughness, 9 , 10 high strength, and good magnetic performance, iron-based amorphous\nalloy films can be used as flexible film sensors to measure changes\nin magnetic signals, which has attracted great attention in wearable\nelectronic devices, 11 electronic skin,\nfood, clinical medicine, and industrial fields (such as complex surfaces). 12 − 16 Based on sensor domain applications, the study of the magnetic surface\neffects of films is a key problem. During the preparation of\niron-based amorphous alloy films, two\nkinds of surfaces with different characteristics are formed. One is\nthe rougher surface with larger surface roughness and many defects,\nsuch as pores, which present weaker superhydrophobic characteristics\nthan the other surface. The other is the smoother surface with lower\nsurface roughness, fewer impurities, and stronger superhydrophobic\ncharacteristics than the rough surface. 7 , 8 , 17 , 18 The surface of iron-based\namorphous films is not only hydrophobic but also oleophobic. 17 At present, the surface modification of\niron-based amorphous alloy\nfilms is based on the rougher surface with weaker superhydrophobic\ncharacteristics, and the chemical conversion membranes are mainly\nprepared by the oxidation method. An endogenous transformation film\ncomposed of Cu 0.86 Fe 2.14 O 4 and Cu 0.86 Fe 2.14 O 4 was generated through the\ntransformation reaction on the surface of the catalyst, which improved\nthe wettability between the amorphous alloy band and the epoxy resin. 17 A kind of film on the surface of an iron-based\namorphous alloy with a thickness of about 1 μm was formed by\nreacting in a mixed weak acid environment at 85 °C for 2 h. These\nsurface treatment technologies, however, are all aimed at the rougher\nsurface. It is difficult to modify the smoother surface with strong\nsuperhydrophobic characteristics. Inspired by the strong anti-oxidant\neffect of tea polyphenols in\ntea, this paper attempts to use environmentally friendly tea polyphenol\ntreatment technology to solve simultaneously the surface modification\nproblem of the rougher surface and the smoother surface of iron-based\namorphous alloy films, which will improve the hydrophilicity of both\nsides of the film to epoxy resin. The results will effectively solve\nthe epoxy resin packaging problem of iron-based amorphous alloy films\nused as soft magnetic materials for electronic and electrical devices.\nAt the same time, it will provide a quick and simple, low-cost, and\nstrong anti-interference scheme for the rapid detection of tea polyphenols\nsensor through magnetic surface effect research."
} | 1,620 |
25029105 | PMC4100744 | pmc | 6,257 | {
"abstract": "Background The prevalence of different biotic processes (limiting similarity, weaker competitor exclusion) and historical contingency due to priority effects are in the focus of ongoing discussions about community assembly and non-random functional trait distributions. Methodology/Principal Findings We experimentally manipulated assembly history in a grassland biodiversity experiment (Jena Experiment) by applying two factorially crossed split-plot treatments to all communities: (i) duration of weeding (never weeded since sowing or cessation of weeding after 3 or 6 years); (ii) seed addition (control vs. seed addition 4 years after sowing). Spontaneous colonization of new species in the control treatment without seed addition increased realized species richness and functional richness (FRic), indicating continuously denser packing of niches. Seed addition resulted in forced colonization and increased realized species richness, FRic, functional evenness (FEve) and functional divergence (FDiv), i.e. higher abundances of species with extreme trait values. Furthermore, the colonization of new species led to a decline in FEve through time, suggesting that weaker competitors were reduced in abundance or excluded. Communities with higher initial species richness or with longer time since cessation of weeding were more restricted in the entry of new species and showed smaller increases in FRic after seed addition than other communities. The two assembly-history treatments caused a divergence of species compositions within communities originally established with the same species. Communities originally established with different species converged in species richness and functional trait composition over time, but remained more distinct in species composition. Conclusions/Significance Contrasting biotic processes (limiting similarity, weaker competitor exclusion) increase functional convergence between communities initially established with different species. Historical contingency with regard to realized species compositions could not be eradicated by cessation of weeding or forced colonization and was still detectable 5 years after application of these treatments, providing evidence for the role of priority effects in community assembly.",
"introduction": "Introduction A deeper insight into the mechanisms which control community assembly is central to understand ecosystem functioning and the maintenance of biodiversity. Functional diversity, i.e. the extent of trait differences among co-occurring species [1] , has been proposed as an important characteristic of biological assemblages. The intimate link between traits and the functioning of organisms [2] suggests that patterns of functional trait distributions within and between communities may provide insights into the operation of non-neutral community assembly rules [3] , [4] . Given a regional species pool with unlimited dispersal, community assembly is often assumed to result from two distinct non-random processes of species sorting: environmental filtering (abiotic filters) and species interactions (biotic filters). Environmental filtering is likely to reduce functional diversity by selecting species with similar ecological characteristics ( = trait convergence) which are able to tolerate the local environment [5] , [6] . Biotic processes may produce different patterns of trait distribution. Niche differentiation through resource partitioning prevents coexisting species from being too similar (i.e. limiting similarity [7] , [8] ) and is expected to select for species with different ecological characteristics ( = trait divergence). However, it also has been discussed that competition may increase similarity among species ( = trait convergence), when species with similar traits compete relatively equally, while excluding weaker competitors with different traits (i.e. equalizing fitness processes [8] , [9] ). This niche-based view of community assembly has been challenged by the “neutral theory” [10] assuming that functional differences do not play a role in community assembly and that the fitness of all species in a community is equivalent. Different community histories, i.e. priority effects due to differences in the initial floristic composition, the order of arrival of new species and their initial establishment, may also impact the outcome of community assembly and prevent convergence in species and functional trait composition under uniform environmental conditions [11] – [13] . In natural ecosystems, the importance of biotic processes causing trait convergence and divergence in community assembly is difficult to disentangle because abiotic processes may also cause trait convergence or counterbalance biotic trait divergence [14] , [15] . In addition, long-term effects due to priority effects and the role of dispersal limitation are hard to identify in communities with unknown assembly history. Biodiversity experiments, where abiotic effects are controlled for by randomizing plots with different plant diversity under relatively homogeneous environmental conditions are an opportunity to study biotic processes of community assembly [16] . In previous experiments it has been shown that, when experimental communities of different species richness are opened to colonization of new species, realized species richness can converge rapidly to similar levels [17] . Convergence can be reached even faster when dispersal limitation is reduced by seed addition [18] . However, convergence of species richness may not be paralleled by convergence of functional trait distribution or species composition and thus provides only limited insight into community assembly. Community-wide use of available niche space encompasses the following components: (i) the amount of niche space spanned out by species in a community (functional richness; FRic), (ii) the evenness of abundance distribution of functionally different species (functional evenness; FEve), and (iii) the degree to which the abundance distribution of functionally different species maximizes divergence in functional trait distributions within a community (functional divergence; FDiv) [19] . In the present study, we used experimental plots of a grassland biodiversity experiment (Jena Experiment [20] ) initially established with different species richness (1, 2, 4, 8, and 16 species) and functional group number and composition (1 to 4) to study community assembly processes in response to two factorially crossed split-plot treatments [21] : (i) duration of weeding leading to different colonization periods of communities (cessation of weeding 6 years after sowing < cessation of weeding 3 years after sowing < never weeded since sowing); (ii) seed addition (control vs. seed addition 4 years after establishment of initial species compositions). During a 5-year study period following the seed addition we monitored species richness and composition and different components of functional diversity (FRic, FEve, FDiv [22] ). We assume that our experimental approach controls for abiotic processes of community assembly and that seed addition removes dispersal limitation. We test the following hypothesis: (1) functional richness (FRic) increases in parallel to realized species richness during colonization due to limiting similarity; (2) increasing functional richness is succeeded by a decline in functional evenness (FEve) and functional divergence (FDiv) due to weaker competitor exclusion; (3) the removal of dispersal limitation due to seed addition increases the chance for the establishment of more species with traits advantageous in competition and weaker competitor exclusion, thus reducing realized species richness and FRic in the longer term; (4) historical contingency, i.e. priority effects due to initial species diversity and longer colonization periods, decelerate species gain and increase in FRic; (5) contrasting biotic processes during community assembly increase similarity in functional trait composition between communities through time, but historical contingency remains visible in distinct realized species compositions of communities initially established with different species richness and composition.",
"discussion": "Discussion In spite of sustained efforts to understand patterns of community assembly, the importance of different biotic processes is poorly disentangled so far. We used the opportunity to study biotic processes of community assembly under relatively homogeneous environmental conditions in split-plots of a large grassland biodiversity experiment opened for colonization by new species and removed dispersal limitation through a seed addition treatment [21] . Species richness and functional trait diversity Species gain and increase in functional richness (FRic) after the opening of experimental communities for colonization by new species was reduced by a higher number of initially established species and a longer colonization period (shorter duration of weeding). The negative effects of a higher initially sown plant diversity on the spontaneous colonization of new species may be attributable to the lower stability of low-diverse communities even in the absence of weeding [40] , while disturbance through weeding and the removal of competitors in communities with a shorter colonization period may have accelerated the colonization success of new species. The long-term effects of higher initial plant diversity in reducing the number of colonizer species are in line with previous short-term studies in the Jena Experiment and other biodiversity experiments (e.g. [41] – [43] ). In our experiment, communities established with a lower number of species even exceeded realized species richness of communities established with a higher number of species after communities were opened for colonization ( Fig. 2A ). Seed addition led to a higher species gain and FRic increase than without seed addition, and the efficiency of seed addition was increased, when weeding was continued for a longer period. Higher levels of realized species richness after seed addition are commonly found because plant communities are often under-saturated with species due to a limited species pool or dispersal constraints [44] , [45] . Although none of the species added as seed failed to establish completely after seed addition [21] and single communities accumulated indeed up to 60 species one or two years after seed addition, average species richness in the seed addition treatments declined again and did not exceed 35 species in the last year of the study ( Fig. 2A ). This upper limit of realized species richness is close to the realized species number in plots initially established with all 60 experimental species in the Jena Experiment (unpubl. data) and indicates that the seed addition led to forced colonization with a short-term over-saturation with species followed by species loss. Functional richness (FRic), which quantifies the volume of functional space filled by a community, increased in parallel to species gain in sub-plots without seed addition, but the increment of FRic was flattened in subplots with a higher number of initially sown species ( Fig. 3B ). The concurrent increase in species richness and FRic indicated that newly colonizing species were functionally different from residents, which is in line with the view that complementary resource requirements of resident and colonizer species select for a maximization of functional richness (indicating limiting similarity). In contrast, FRic reached maximum values in the year after the seed addition in sub-plots with seed addition ( Fig. 3C ), followed by maximum values of functional divergence (FDiv) in the third year ( Fig. 3I ) before both measures of functional trait diversity declined more strongly than realized species richness in subsequent years. Functional evenness was also higher after seed addition, but decreased continuously through time ( Fig. 3F ). Decreasing FEve in parallel to species gain ( Fig. 3E ) and a minor trend in FDiv ( Fig. 3H ) through time, however, occurred in subplots without seed addition. FDiv describes how abundance is spread along functional trait axes, where large values of FDiv are expected to indicate a high degree of niche differentiation [19] . Our analysis of functional trait diversity was based on multiple traits. Although different functional traits may be associated with different processes during community assembly and relate to different niche axes [46] , [47] , plants have to balance different functions which is often reflected in fundamental functional trade-offs. Probably, the short-term increase in FDiv and maximum levels of FRic after seed addition were due to the advantage of species with traits associated with rapid establishment followed by weaker competitor exclusion. Weaker competitor exclusion (related to equalizing fitness processes) in grasslands at high productivity levels is discussed as a biotic process resulting in niche reduction in favour of perennial species with higher ability for resource competition [6] , [48] , [49] . Decreasing FEve may be either due to abundances less evenly distributed among species or to less regular functional distances among species [22] . The decline in FEve through time as well as a reduced FEve in subplots with a longer colonization period ( Fig. 4D–F ) indicated again that equalizing fitness processes favoured species with more similar trait combinations during community assembly. Convergence in species and functional trait composition Previous studies have reported that experimental communities initially sown with different plant diversity and opened to colonization converged in species richness and functional or phylogenetic composition while keeping different species composition [13] , [50] . Our results based on a 5-year study period starting 4 years after establishing communities with different plant diversity also showed stronger between-community convergence in functional trait composition than in species composition. In subplots with a different assembly history, however, we found a divergence in communities initially established with higher plant diversity (4 and more species, Fig. 5B ), while communities initially established as monocultures or 2-species mixtures maintained their initial convergent state. Probably, this divergence at higher species-richness levels was due to the stochasticity in colonization processes and the persistence of priority effects, which may cause high variability in community structure among similar sites [51] . It becomes increasingly apparent that multiple processes are involved in community assembly. A number of recent studies have shown that patterns of functional trait composition depend on environmental conditions and the traits considered [46] , [48] , [49] , [52] . Our study based on multiple traits provided evidence that contrasting biotic processes govern shifts of functional community composition in experimental grasslands with relatively homogeneous environmental conditions. Limiting similarity played a larger role during early phases of colonization. Colonization of new species went in parallel with an increase in FRic indicating that the newly colonizing species were functionally different from residents (hypothesis 1). Increasing FRic was succeeded by a decline in FEve suggesting weaker competitor exclusion in the longer run (hypothesis 2). These processes were accelerated when seed limitation was removed through seed addition resulting in peak values of FRic and larger values of FEve, which declined again in subsequent years (hypothesis 3). Nevertheless, FRic and FEve remained at higher levels in subplots with seed addition compared to those without seed addition during the 5-year study period, indicating that the removal of seed limitation improved the chance for assembling communities with a higher number of species with traits advantageous in competition. However, in addition to the series of assembly processes related to limiting similarity and weaker competitor exclusion, our study has clearly shown that historical contingency in assembly processes due to priority effects may be traceable over longer time (hypothesis 4). First, species gain and increase in FRic were considerably reduced when plant communities were initially established with greater diversity such that, realized species richness and FRic of initially low-diverse communities were even greater than in initially high-diverse communities after opening for colonization. Second, a longer duration of the colonization period decreased the accelerated species gain and the increase in FRic in initially low-diverse communities and maintained FEve at lower levels. In addition, the prominent role of priority effects becomes visible by the lagged convergence of species composition relative to functional trait composition of communities initially established with different species compositions (hypothesis 5)."
} | 4,265 |
30209344 | PMC6330079 | pmc | 6,258 | {
"abstract": "Horizontally acquired genes can be costly to express even if they encode useful traits, such as antibiotic resistance. We previously showed that when selected with tetracycline, Escherichia coli carrying the tetracycline-resistance plasmid RK2 evolved mutations on both replicons that together provided increased tetracycline resistance at reduced cost. Here we investigate the temporal dynamics of this intragenomic coevolution. Using genome sequencing we show that the order of adaptive mutations was highly repeatable across three independently evolving populations. Each population first gained a chromosomal mutation in ompF which shortened lag phase and increased tetracycline resistance. This was followed by mutations impairing the plasmid-encoded tetracycline efflux pump, and finally, additional resistance-associated chromosomal mutations. Thus, reducing the cost of the horizontally acquired tetracycline resistance was contingent on first evolving a degree of chromosomally encoded resistance. We conclude therefore that the trajectory of bacteria-plasmid coevolution was constrained to a single repeatable path."
} | 282 |
34818037 | PMC8612681 | pmc | 6,260 | {
"abstract": "Multicomponent self-assemblies cross-regulate their structures by exchange of molecular information using a metabolic network.",
"introduction": "INTRODUCTION Complex biological systems can sense and respond to external stimuli and can cross-regulate each other by principles of communication using molecular messengers. This leads to a broad ensemble of spatiotemporally controlled, communicating and self-regulating structures that are orchestrated through signaling systems and feedback loops ( 1 , 2 ). Such biological reaction networks can be mimicked using chemical reaction networks (CRNs) that show increasingly complex behavior and which have been coupled with self-assembling building blocks to make transient systems of single species ( 3 – 6 ). However, realizing more elaborate behavior, such as communication and cross-regulation of different entities, remains a challenge ( 2 , 7 ). One promising approach toward versatile CRNs is isothermal DNA strand displacement (DSD), in which short complementary domains, termed toeholds, allow for programming of complex reaction networks ( 8 ) able to sense ( 9 ), communicate ( 10 ), and execute simple logic gates ( 11 , 12 ), as well as compute logic circuits ( 13 , 14 ). The behavior of DSD circuits is programmable in a straightforward manner because of the Watson-Crick base pairing of DNA and can be predicted using thermodynamic and kinetic models ( 12 , 15 , 16 ). Several programs ( 14 , 17 – 20 ), such as Visual DSD ( 21 – 23 ), have been developed for this purpose. The information processing of DSD circuits complements both biological and chemical systems for a wide range of applications ( 24 , 25 ). For example, de Greef and co-workers ( 10 ) showed stable DNA circuits performing versatile functions in protein-based protocell arrays, giving rise to autonomous sensors with cell-like behavior. Recently, first efforts coupled DSD cascades with structural DNA nanotechnology. For instance, Winfree and co-workers ( 26 ) used DSD circuits with an amplifier to control the self-assembly (SA) of small DNA tiles, Franco and co-workers ( 27 ) showed transient DNA fibrils based on a genelet on/off switch with resulting transcriptional control, and Woods et al. ( 28 ) executed Boolean circuits using DNA tiles and a DNA origami seed. In addition, DSD cascades can also be interfaced with adenosine 5′-triphosphate (ATP)–driven CRNs to create previously unknown behavior, such as self-resetting DSD systems, that again can be interfaced with multicomponent structural assembly ( 29 ). These and other studies ( 30 , 31 ) give a glimpse on the immense capacity of combining structural and dynamic DNA nanotechnology to pioneer new adaptive and autonomous biosynthetic materials and devices. Among the multitude of DNA-functional or DNA-active building blocks, DNA origamis, obtained by folding a long DNA scaffold strand into distinct shapes with short staple strands ( 32 – 35 ), are a prime candidate for integrating DSD circuits with structural DNA nanotechnology because of their capabilities for distinct superstructure formation and precise stoichiometric functionalization. Toehold-mediated strand displacement has been used to reconfigure finite DNA origami superstructures ( 36 – 38 ), and even first logic gates have been implemented ( 39 ). We previously showed periodic supracolloidal DNA origami fibrils both by double-stranded DNA (dsDNA) hybridization ( 40 ) and host/guest interaction with programmable multivalent interactions ( 41 , 42 ), and introduced reversible DNA origami nanotube formation responding to single-stranded DNA (ssDNA) fuel/antifuel triggers ( 43 ). Here, we introduce modular DSD circuits of increasing functionality to program the cross-regulation of two fibril-forming three-dimensional (3D) DNA origami species in an autonomous preprogrammed system via communication relays integrated into the origami species. The DSD circuits control the sorted origami polymerization in time and allow the two distinct origami building blocks to communicate with each other via ssDNA signals. The two DNA species used are (i) 3D DNA origami hollow nanocylinders and (ii) 3D DNA origami nanocuboids, which self-assemble into fibrillar structures depending on the availability of signals. The DSD circuits first realize a negative feedback loop that is subsequently extended by an amplifier module, which enhances the negative feedback, and, lastly, with a thresholding gate to impose additional temporal delays in the signal transduction process. The DSD circuits are based on an in silico design, established with simple ssDNA systems, and subsequently extended to DNA origami. Experimentally, we study the systems using a combination of a in situ Förster resonance energy transfer (FRET) readout and ex situ transmission electron microscopy (TEM). The colloidal level provided by the use of 3D DNA origami will be of interest for future modification with, e.g., enzymes or inorganic nanoparticles. In addition, while communication between species and coupled behavior has been shown on a molecular basis for DNA, this needs to be coupled to changes on the mesoscale to truly mimic biological behavior inspired from biological filaments based on structural proteins, and to transfer these learnings to practical applications in mesoscopic materials at some point. We suggest that this versatile and modular approach for merging structural and dynamic DNA nanotechnology furthers understanding of dynamic soft multicomponent systems while opening up new possibilities for DNA origami superstructures, emulating behavior of biomimetic filaments such as microtubules, which have shown cross-regulation with actin filaments ( 44 ).",
"discussion": "DISCUSSION We introduced the first example for autonomous cross-regulation of self-sorting multicomponent self-assembling systems by concatenating DSD scenarios with molecular information relays embedded in the self-assembling species. The approach merges structural DNA nanotechnology in the form of precisely programmable 3D DNA origami with a DSD-CRN to precisely orchestrate the reconfiguration of superstructures proceeding via an intermediate 3D DNA origami colloid level. The colloidal 3D nature facilitates distinction in microscopy and mimics globular protein structures for biomimetic filament design. Even though on a rudimentary level, the fibrillar structures mimic some behavior of the living cytoskeleton, where various fibrillar structures such as microtubules and actin filaments cross-regulate ( 44 ) and are organized temporally. Because of the versatility of the DSD system, the basic negative feedback module could be extended with amplification and thresholding modules, and the in silico optimization provides useful guidance for experimental design and analysis. The amplification module further allows to change the end point state (fibril or monomer) for the origami cuboids to synchronize the transient behavior with the origami cylinder nanotubes. Gratifyingly, both origami building blocks are easily distinguishable by TEM, allowing to quantify the respective fibril length distribution throughout the course of the DSD circuit, a distinct advantage of this system compared to DNA tile–based approaches. Next to the temporal features of the DSD circuit, both the origami concentration and the connector density influence the fibril lengths. Compared to pure strands in solution, the origami system experiences delays in the operation of the circuit because of the spatial confinement at the origami tips; yet, they continue to operate smoothly despite the higher level of confinement and higher dimensionality of the spatially immobilized sequences: 2D in 3D DNA origami versus 1D in 2D sheet origami or molecular scale for DNA tiles. The DSD circuit presented herein provides a viable starting point to further extend the behavior by, for instance, including catalytic DSD cycles, ATP-driven DSD cycles, or even incorporate additional external switches and signal processors ( 29 ). First examples on DNA tile–based fibrils have shown fascinating approaches for redox ( 55 ) or pH ( 56 ) controlled polymerization, and combining these approaches both with DNA origami as colloidal monomers and with DSD computing is promising for sensing and even biomedical applications. In addition, the elaborate design of the multivalency patterns at the interacting tips can offer broad opportunities including pattern recognition ( 42 ). We also suggest that the control principles should be applicable to other colloidal structures, which can allow, at the least, the organization of relevant photonic effects in structured matter ( 57 )."
} | 2,167 |
30534351 | PMC6280393 | pmc | 6,261 | {
"abstract": "Rhizobia bacteria engage in nitrogen-fixing root nodule symbiosis, a mutualistic interaction with legume plants in which a bidirectional nutrient exchange takes place. Occasionally, this interaction is suboptimal resulting in the formation of ineffective nodules in which little or no atmospheric nitrogen fixation occurs. Rhizobium leguminosarum Norway induces ineffective nodules in a wide range of Lotus hosts. To investigate the basis of this phenotype, we sequenced the complete genome of Rl Norway and compared it to the genome of the closely related strain R. leguminosarum bv. viciae 3841. The genome comprises 7,788,085 bp, distributed on a circular chromosome containing 63% of the genomic information and five large circular plasmids. The functionally classified bacterial gene set is distributed evenly among all replicons. All symbiotic genes ( nod , fix , nif ) are located on the pRLN3 plasmid. Whole genome comparisons revealed differences in the metabolic repertoire and in protein secretion systems, but not in classical symbiotic genes. Electronic supplementary material The online version of this article (10.1186/s40793-018-0336-9) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions Although detrimental in agriculture, ineffective nitrogen-fixing symbiosis remains poorly investigated. In this regard, Rl Norway is an interesting strain as it exhibits a parasitic behaviour in a wide range of hosts. Comparative genomic analyses with other R. leguminosarum strains have the potential to reveal novel factors mediating symbiotic compatibility and efficiency.",
"introduction": "Introduction Legume crops are central to sustainable agricultural practices and food security [ 1 , 2 ]. They have a low need for synthetic nitrogen fertilizers input, as they engage in a symbiosis with a group of diazotrophic bacteria collectively known as rhizobia. This symbiotic interaction is initiated by a molecular crosstalk between rhizobia and their cognate legume host. Upon recognition of specific signals, legume plants intracellularly accommodate rhizobia inside root organs called nodules, where they engage in a bidirectional nutrient exchange [ 3 ]. Occasionally, suboptimal interactions establish between the symbiotic partners. These lead to the formation of ineffective nodules in which limited to no nitrogen fixation occurs. These ineffective symbiotic associations are characterized by the formation of small white nodules, which results in reduced or no plant growth promotion [ 4 ]. Ineffective nitrogen-fixing symbioses have been described after introduction of crop legumes into areas where previously native legumes grew. The soil microbiota associated to native species can often outcompete inoculant strains [ 5 ]. For instance, ineffective nitrogen fixation occurs in fields where perennial and annual clovers co-exist [ 6 , 7 ]. In field trials, inoculant strains were unable to completely overcome indigenous R. leguminosarum bv. trifolii strains and occupied on average 50% of the nodules [ 8 ]. In extreme cases, it has been shown that endogenous rhizobia can completely block the nodulation of introduced rhizobia. For example, the nodulation of pea cultivars Afghanistan and Iran by rhizobial inoculants is suppressed in natural soils by the presence of a non-nodulating strain [ 9 ]. However, although ineffective nodulation is a limiting factor for sustainable agriculture, the molecular basis underlying it remains largely unknown [ 10 ]. Rhizobium leguminosarum ( Rl ) strains are cognate micro-symbionts of legumes, including Pisum , Lens , Lathyrus , Vicia , Phaseolus and Trifolium [ 11 ]. However, a R. leguminosarum strain isolated from a Lotus corniculatus nodule in Norway exhibits a different compatibility range that includes several Lotus species and ecotypes. Rl Norway does not induce effective nodules in any Lotus species tested so far [ 12 ]. Strikingly, there are host genotype specific differences in the nodulation phenotypes induced by Rl Norway, as it cannot induce nodules on L. japonicus Gifu, but induces bumps on L. japonicus Nepal, and white nodules on L. burttii and L. japonicus MG-20. This is in contrast to compatible Mesorhizobium strains that induce monomorphic phenotypes in the same plant ecotypes [ 12 ]. The striking diversity of ineffective nodulation phenotypes induced by Rl Norway in Lotus motivated us to sequence and annotate its complete genome, and to compare it to the published genome of R. leguminosarum bv. viciae 3841 ( Rlv 3841), a well-characterised R. leguminosarum strain. Here, we show that the genomes are largely conserved. There are no major differences in the nif and fix clusters required for nitrogen fixation and in the nod cluster essential for the production of nodulation factor. However, differences were observed in terms of metabolic and protein secretion system genes."
} | 1,238 |
35512445 | PMC9440436 | pmc | 6,262 | {
"abstract": "Abstract Nitrogen (N) fixation in cereals by root-associated bacteria is a promising solution for reducing use of chemical N fertilizers in agriculture. However, plant and bacterial responses are unpredictable across environments. We hypothesized that cereal responses to N-fixing bacteria are dynamic, depending on N supply and time. To quantify the dynamics, a gnotobiotic, fabricated ecosystem (EcoFAB) was adapted to analyse N mass balance, to image shoot and root growth, and to measure gene expression of Brachypodium distachyon inoculated with the N-fixing bacterium Herbaspirillum seropedicae . Phenotyping throughput of EcoFAB-N was 25–30 plants h −1 with open software and imaging systems. Herbaspirillum seropedicae inoculation of B. distachyon shifted root and shoot growth, nitrate versus ammonium uptake, and gene expression with time; directions and magnitude depended on N availability. Primary roots were longer and root hairs shorter regardless of N, with stronger changes at low N. At higher N, H. seropedicae provided 11% of the total plant N that came from sources other than the seed or the nutrient solution. The time-resolved phenotypic and molecular data point to distinct modes of action: at 5 mM NH 4 NO 3 the benefit appears through N fixation, while at 0.5 mM NH 4 NO 3 the mechanism appears to be plant physiological, with H. seropedicae promoting uptake of N from the root medium.Future work could fine-tune plant and root-associated microorganisms to growth and nutrient dynamics.",
"conclusion": "Conclusion Improvement of plant performance through the use of beneficial microbes is dynamic and thus must include understanding of the interaction through the plant’s developmental period under varying abiotic conditions including fertilizer supply. The adaptations to the gnotobiotic EcoFAB-N chamber in this study allow its application to nitrogen-related experiments with the use of N-fixing bacteria, and can be expanded to other nutrient conditions. The non-invasive phenotyping pipeline allows precise monitoring of root and shoot parameters through time, including analysis of primary and lateral root lengths and root hairs, which are not possible using simple end-point measurements. Finally, the approach for the mass-balance calculation is transferable to multiple nutrients and potentially beneficial organisms including P from algal biomass ( Mau et al. , 2021 ). In this particular case we saw promotive action by H. seropedicae on B. distachyon growth at high and low NH 4 NO 3 . The time-resolved phenotypic and molecular data, however, point to distinct modes of action: at 5 mM the benefit appears through N fixation, while at 0.5 mM the mechanism appears to be plant physiological, with H. seropedicae promoting uptake of N from the root medium.",
"introduction": "Introduction Nitrogen (N) fertilizer use has been increasing since the industrial revolution, reaching 145.5 million metric tons worldwide in 2018 ( www.fertilizerseurope.com ). In some countries laws have placed stringent limits on the application of N fertilizers, with the aims of minimizing the leaching that leads to eutrophication of freshwater resources, decreasing environmental impact of fertilizer production ( Bundesministerium der Justiz und für Verbraucherschutz, 2017 ), and reducing emissions into the atmosphere and climate warming. To minimize synthetic fertilizer use but sustain and improve plant performance to meet population demand, the following are required (i) increased N 2 fixation from the atmosphere and N uptake into the root through N-fixing bacteria ( Glick, 2010 , 2012 ), and (ii) optimized use of N within the plant. The cereals wheat, maize, and rice represent 70% of global crops. In cereals, the global use efficiency of nitrogen fertilizer increased only 2% in the 13 years between 2002 and 2015, from 33% to 35%. The gain is attributed to agronomy using precision agriculture and breeding of more N-efficient varieties ( Omara et al. , 2019 ). Nitrogen is taken up by cereal roots through a system of transporters on the root cell plasma membrane with high or low affinity for N-containing ions (ammonium and nitrate), and the activation of each system is dependent on the environmental conditions ( O’Brien et al. , 2016 ). Notably, plant roots exist in a dynamic environment, with temporal changes in growth and anatomy, expression of nutrient transporters that is root type and age dependent, and constant interaction with soil and microorganisms of varying quality ( Arsova et al. , 2020 ). Root-associated bacteria that live within or on the roots in the rhizosphere are increasingly being developed for commercial use in cereal agriculture ( de Souza et al. , 2015 ). However, the exact mode of action is often not understood ( Dent and Cocking, 2017 ) and performance in field crops is unpredictable and inconsistent ( Pii et al ., 2015 ), with growth effects ranging from negative to positive across global wheat trials. To fully exploit the potential of N-fixing root-associated bacteria, we need to understand the developmental and molecular modes of action of members of the grass family, Poaceae , over time. We use the model grass Brachypodium dystachion , with similar but smaller genome ( Brkljacic et al. , 2011 ), similar root development ( Watt et al. , 2009 ), and similar microbiome ( Kawasaki et al. , 2016 ) to cereals, along with Herbaspirillum seropedicae , an endophytic diazotroph that has been shown to colonize B. distachyon ( do Amaral et al. , 2016 ). Herbaspirillum spp. are found in roots, stems, and leaves of many plants, most without disease symptoms ( Roncato-Maccari et al. , 2003 ), and can promote the growth of Poaceae (maize, Michantus , rice, sorghum, and sugarcane; Monteiro et al. , 2012 ). Specifically, H. seropedicae increased growth and nitrogen accumulation in rice varieties ( Divan-Baldani et al. , 2000 ; Gyaneshwar et al. , 2002 ; Roncato-Maccari et al. , 2003 ; Trovero et al. , 2018 ). In light of the variable plant growth-promoting rhizobacterial response found in the field, we hypothesized that B. distachyon ’s response to H. seropedicae will be dynamic, will vary depending on the environment the plants are grown in (in our case with varying N availability), and in addition, will change through time. A recently established fabricated ecosystem, EcoFAB ( Gao et al. , 2018 ), joins a palette of microfluidic devices developed to study plant response to various conditions ( Massalha et al. , 2017 ). Importantly, EcoFAB was shown to be a reproducible system in a ring-trial that followed not only phenotypic but also metabolomic traits across four laboratories on two continents ( Sasse et al. , 2019 ). EcoFABs remain gnotobiotic over time, with reproducible B. distachyon root and shoot phenotypes under different P supply ( Sasse et al. , 2019 ). To quantify and resolve plant–microorganism dynamics in the B. distachyon – H. seropedicae phenotype, we modified the EcoFAB to supply different N levels to the plant (from here on EcoFAB-N), and quantified N uptake and imaged roots and shoots non-invasively over time. Non-invasive phenotyping allows the acquisition of temporal and spatial plant parameters from the time microorganisms are introduced to a system until a pre-determined point in plant growth ( Schillaci et al , 2021 ). Non-invasive plant phenotyping has been rapidly extended to include mobile, inexpensive, and portable methods, such as use of mobile phones ( Müller-Linow et al. , 2019 ), promoting applicability in labs around the world. So, our second hypothesis states that a gnotobiotic system in combination with non-invasive imaging has the potential to achieve temporal resolution of the plant response to microbes, on the tissue, molecular, and nutritional levels. Thus, we combined non-invasive measures with uptake of different N forms by plants from the root medium, with repeated biomass and N sampling and transcript analysis at two destructive time points, to resolve the dynamics of the plant–bacterial phenotype for N fixation in cereals.",
"discussion": "Discussion The EcoFAB-N coupled to whole plant imaging and N mass-balance analysis revealed a dynamic H. seropedicae – B. distachyon phenotype. N availability and time were the most important variables, and root and leaf responses varied in magnitude and direction. Further, inoculation promoted primary root growth, but inhibited root hair length, irrespective of nitrogen availability and time, while nitrogen form (nitrate or ammonium) and potentially N source (medium or bacterial) also varied. The main findings of the study are summarized in Fig. 9 . Dynamics are important to consider when interpreting variable responses to N-fixing bacteria in cereals in agriculture. Fig 9. Summary of the response of B. distachyon to inoculation with H. seropedicae (HS) in two nitrogen conditions. At 0.5 mM NH 4 NO 3 HS causes increase of leaf area, elongation of primary root, and shortening of root hairs. Inoculated plants also depleted NH 4 + faster from the medium. Their nitrate uptake genes NRT2.1 (both time points) and NRT1.1 (28 DAT) in shoot have higher expression, corresponding to behavior at low NO 3 − . At 5 mM NH 4 NO 3 inoculated plants show longer primary roots and shorter root hairs than non-inoculated plants. They deplete less NO 3 − from the medium. Yellow stars indicate significant differences by non-invasive phenotyping. Relative expression, as ratio of inoculated versus non-inoculated is shown in the heat maps, with high (dark blue) and low ratio (white); asterisks indicate significant t -test in the inoculated versus non-inoculated plants. N-form uptake (gray triangles): increased depletion from medium (upright traingle), decreased depletion (inverted triangle). Control, non-inoculated; +HS, inoculated plants; LR, lateral roots; PR, primary root; RH, root hairs. EcoFAB-N and the non-invasive phenotyping pipeline are flexible and allow further expansion by the scientific community One big advantage of the open source EcoFAB platform is that the chamber is adjustable and can therefore be configured based on the experimental needs, e.g. nutrition and aeration in the case of aerobic bacterial interactions (this work, EcoFAB-N), or plant space requirements ( Gao et al. , 2018 ). We show here that the closed EcoFAB-N system provides an accurate method to study mass balance, such as N mass balance investigated in this study, which is challenging in natural systems. Mass balance analyses coupled to time-resolved phenotypic data are a useful way to estimate nutrients taken up by plants growing with complex biotic and abiotic nutrient forms ( Mau et al. , 2021 ). By extending the EcoFAB platform to include non-invasive shoot and root imaging, we increased the throughput of growth phenotyping to a turnover of 25–30 plants h −1 . The EcoFAB-N pipeline allows study of the whole plant growth and N fluxes while maintaining the gnotobiotic environment, with integration of molecular responses within the dynamic phenotype at specific time points, via invasive harvests. We found that B. distachyon grown in high N medium had greater leaf area and shoot biomass, and comparable root length to B. distachyon grown at lower N. This fits with expected plant behavior under N limitation ( Cambui et al. , 2011 ; David et al. , 2019 ). The resource allocation in the case of the high 5 mM NH 4 NO 3 was greater towards shoots, compared with in the case of 0.5 mM NH 4 NO 3 , which was directed to the root system ( Fig. 3C ). The results from the EcoFAB-N also aligned well to wheat responses to similar N levels when grown in paper pouches ( Tolley and Mohammadi, 2020 ). Additionally, we observed a root hair response that was linked to N supply. Here we observe shorter root hairs at 5 mM NH 4 NO 3 than at 0.5 mM NH 4 NO 3 , although newly produced root hairs were shortening through time. While root hairs have been shown to respond to P deficiency and influence uptake, information about N is not as clear. However, in tomato, certain ammonium and nitrate transporters are expressed in the root hairs, suggesting root hair cells may have a role in N uptake ( Gilroy and Jones, 2000 ). Studies of wild grasses reported shorter root hairs with increased N availability ( Boot and Mensink, 1990 ). Recently a modeling approach indicated that root hairs are important during N deficiency ( Saengwilai et al. , 2021 ). We conclude that root hair length is a trait worthy of further investigation for more N-efficient varieties, and that plant development is an important variable for full understanding of the contribution of length. The phenotypic and transcript responses of B. distachyon to H. seropedicae differed depending on the nutritional conditions and time Our results lead us to the conclusion that B. distachyon responses to H. seropedicae inoculation are strongly dependent on the nutrient condition where the interaction takes place. We observed two types of response in inoculated plants. Firstly, conserved responses to H. seropedicae inoculation, like increase of total root length or decrease of root hair length, irrespective of the available N ( Fig. 3B , F ). The differences here can be described as a cumulative response to N availability and H. seropedicae inoculation. The second type of response was an N-related response to H. seropedicae in inoculated plants. For example, H. seropedicae -induced change in leaf area was increased only in the low N condition ( Fig. 3A ). Closer observation showed that several measured parameters fit into this group by showing different response to H. seropedicae inoculation in 5 mM and 0.5 mM NH 4 NO 3 (i.e. leaf area, the depletion of NH 4 + and NO 3 − , as well as expression of AMT1.1 and NiR at 28 DAT, Figs 3A , 4C , 7D , F ). Time, both as a factor in plant development and from the moment of inoculation, also played a role in plant–bacterial dynamics, in addition to nutrient supply, consistent with Schillaci et al. , (2021) . For example, at 11 DAT, total root length ( Fig. 3B ) increased in inoculated plants at 0.5 mM NH 4 NO 3 , but at 28 DAT total root length increase was only measured in the 5 mM NH 4 NO 3 treatment. We observed differences in the NH 4 + and NO 3 − depletion at different times as well, in inoculated and non-inoculated plants, and in both conditions ( Fig. 4A , B ), with decreased NH 4 + and NO 3 − depletion (lower uptake by the plant) in the 5 mM condition at the start of the time series (8 and 11 DAT) and slightly increased depletion (higher uptake) in the 0.5 mM condition towards the end of the time series. The molecular components in the N uptake system also varied through time ( Figs 7 , 8 ). The expected behavior in fully developed plants is that the AMT and NRT transporter genes, as well as enzymes, are more highly expressed at lower NH 4 + and NO 3 − concentrations ( David et al. , 2019 ). While we often saw this behavior (e.g. GS , Figs 7A , B , 8G ), we saw deviations as well. Notably most studies investigate plant responses at a specific time in development, but plant development including utilization of seed resources needs to be considered for proper understanding of the dynamic transcript expression ( Enns et al. , 2006 ). We speculate that part of the N-related response was related to resources in the seed, and the amount impacts the expression of the various transcripts measured in this study. Particularly in roots, we saw a large variation among the monitored nitrogen transporters in this study, whereas the behavior of the central metabolic enzymes appeared to be more stable. A longer time series may lead to a better quantification of molecular responses in the future studies, but the data presented here already show similar NR (at 20 and 28 DAT; Fig. 7E ; Supplementary Fig. S4A ) and NiR (28 DAT; Fig. 7F ) expression. In inoculated plants, NiR , whose protein reduces NO 3 − to NH 4 + , had lower transcript expression at 28 DAT in the 0.5 mM condition ( Fig. 7F ). The preceding enzyme in the NO 3 − reduction pathway responded similarly ( NR ; Supplementary Fig. S4A ) to H. seropedicae indicating that this is a robust response to the bacterium. The general expression pattern (lower expression at low N, higher in high N) was also followed by the nitrate transporter gene NRT2.1 . Interestingly the expression of the cytosolic glutamine synthetase gene ( GC1.1c ; Fig 7A , B ), involved in NH 4 + metabolism, in B. distachyon was not altered by H. seropedicae ; and transcript expression pattern was opposite to the nitrate-related enzymes (higher expression in low N conditions compared with high N). This leads to two questions: does B. distachyon have a preference for ammonium rather than nitrate? Do specific transcripts respond to the presence of a given bacterial inoculant? Future EcoFAB-N experiments with KNO 3 and (NH 4 ) 2 SO 4 as sole nitrogen sources combined with phenotyping and extensive sequencing will be needed to answer these questions. \n H. seropedicae inoculation altered N mass balance of B. distachyon in EcoFAB-N Although N-fixation by free-living bacteria associated with grasses provides a lower amount of N to crops compared with nitrogen provided from Rhizobium spp. to leguminous plants ( Rosenblueth et al. , 2018 ), a small contribution to grasses is important owing to the large global cereal agriculture. In this study, B. distachyon plants grown for 28 DAT supplied at 5 mM NH 4 NO 3 contained 11.34% N that could not be accounted for based on medium uptake (calculated as in Fig 6 , black bar as percentage of total plant N at 28 DAT, Supplementary Fig. S3 ). The current working hypothesis is that this was provided through the activity of H. seropedicae. Interestingly we did not find any obvious contribution of H. seropedicae to the N content in plants grown on 0.5 mM NH 4 NO 3 . Future experiments using H. seropedicae mutant strains without N-fixation genes, or a change of the 15 N composition in the plant, could be used to confirm the contribution of the bacterium through N fixation. Bacterial gene expression studies at the two conditions could further clarify the bacterial variability and adjustment to the interaction at various N levels. Besides the nitrogen fixation, H. seropedicae might also stimulate N uptake from soil/medium, which has not been intensively investigated. Ribaudo et al. (2006) reported that H. seropedicae inoculation of maize positively affected activities of ammonium assimilation enzymes ( Ribaudo et al. , 2006 ). In this study, we observed increased ammonium and nitrate depletion only in the medium at 0.5 mM NH 4 NO 3 . We thus hypothesize that the rapid N depletion under low N conditions may be stimulated by H. seropedicae . Looking forward, this study stimulates plant-specific questions: Does B. distachyon have a preferred N-form? Do root hairs express transporters for N uptake and could this knowledge be exploited for plants with improved N uptake? Finally, what is the best time for bacterial inoculation in the context of plant developmental stage, and which environmental prerequisites exist for a beneficial plant–microbe interaction in the field? Conclusion Improvement of plant performance through the use of beneficial microbes is dynamic and thus must include understanding of the interaction through the plant’s developmental period under varying abiotic conditions including fertilizer supply. The adaptations to the gnotobiotic EcoFAB-N chamber in this study allow its application to nitrogen-related experiments with the use of N-fixing bacteria, and can be expanded to other nutrient conditions. The non-invasive phenotyping pipeline allows precise monitoring of root and shoot parameters through time, including analysis of primary and lateral root lengths and root hairs, which are not possible using simple end-point measurements. Finally, the approach for the mass-balance calculation is transferable to multiple nutrients and potentially beneficial organisms including P from algal biomass ( Mau et al. , 2021 ). In this particular case we saw promotive action by H. seropedicae on B. distachyon growth at high and low NH 4 NO 3 . The time-resolved phenotypic and molecular data, however, point to distinct modes of action: at 5 mM the benefit appears through N fixation, while at 0.5 mM the mechanism appears to be plant physiological, with H. seropedicae promoting uptake of N from the root medium."
} | 5,164 |
35333041 | PMC9007417 | pmc | 6,264 | {
"abstract": "Friction\ncontinues to account for the bulk of energy losses in\nmechanical systems, with an estimated 23% of the world’s total\nenergy consumption used to overcome friction. Concentrated polymer\nbrushes (CPBs) have recently attracted significant scientific and\nindustrial attention, given their ability to achieve superlubricity\n(i.e., coefficients of friction below 0.01); however, understanding\nthe mechanistic interactions underlying their wear performance has\nbeen largely overlooked. Herein, we employ a custom-built optical\ntest apparatus to investigate the inter-dependencies between CPBs\nand laser-produced surface texture (LST), assessing for the first\ntime the friction, film thickness, and wear behavior in situ and simultaneously.\nRecent developments in picosecond laser etching allowed us to graft\nCPBs atop the finest laser-etched matrix of micron-sized dimples reported\nin literature to date. At low sliding speeds, combined CPB–LST\nreduces the coefficient of friction to 0.0006, while increasing the\nCPB durability by up to 34% through a lateral support mechanism offered\nby the textured micro-features. Furthermore, the imaging results shed\nlight on CPB failure mechanisms. Both these mechanisms of lateral\nsupport and failure propagation impact the wear resistance of CPBs\nand are important in the development of CPBs for future applications\n(e.g., in low-speed bearings functioning under controlled abrasive\nwear conditions).",
"conclusion": "5 Conclusions The ability of the grafted CPB\nto reduce friction and wear in sliding\nbearings is the focus of intense research. However, most studies to\ndate were performed under mild or moderate test conditions, where\npolymer brushes were grafted onto the moving surface of a conformal\ncontact (i.e. , low contact pressures). To understand\nthe impact of CPBs, and their enhancement from LST, under severe contact\nconditions, we used a non-conformal contact subjected to sliding speeds\nup to 2000 mm/s and pressures of 25 MPa. Measurements of friction,\nfilm thickness, and wear response (all\nobtained in situ and simultaneously) were performed using a high-speed,\ndual interferometry technique coupled to a custom-built, ball-on-disc\ntest apparatus. Various textured patterns of pockets with different\ndepths and diameters were compared against their corresponding non-textured,\nCPB-coated, and non-CPB coated references and yielded the following\nconclusions: Observed transient\nfilm thickness reduction at a constant\nsliding speed is caused by hydrodynamic squeeze flow of the IL as\nthe countersurface approaches the compliant CPB surface. Here, the\nconcomitant increase in the shear rate increases friction due to Newtonian\nviscous shear losses. At low sliding\nspeeds (corresponding to short distance\nperformance), the combined CPB–LST approach leads to average\nfriction reductions of more than 99%. Pocketed surfaces are beneficial in terms of wear probably\ndue to the lateral support that the polymer brushes situated inside\nthe textured features (i.e. , the “reinforced\ntough layer”) offer to those grafted outside the pockets (i.e.,\nthe “concentrated bulk layer”) . Shallower pockets reduce friction to the\nmost and offer\nincreased lateral support for the polymer brushes grafted onto the\ndisc surface and thus increase durability. This agrees with results\nobtained using one of the test samples with exceptional material pileup\naround the pocket edges, which were shown to provide additional, artificial\nlateral support and result in the most significant reduction in friction. At sliding speeds greater than 60 mm/s,\nCPB layers are\nremoved instantly. This may be attributed to one or a combination\nof drivers (including sliding distance, shear stress, lifetime of\nthe polymer brushes’ adhesion points, IL squeeze flow, and\nstress concentration-driven peeling), which completely removes the\nCPBs from the contact. Wear of CPB on\ntextured surfaces initiates and gradually\nprogresses along the pocket-free zone located in the center of the\ncontact. However, in non-textured samples, abrasive wear particles\nact to immediately collapse the CPB layer; this suggests that pockets\nsuppress the propagation of CPB failure through additional lateral\nsupport offered to the surface and bulk layers. Carefully selected surface texture can increase the\ndurability of the CPBs layer by up to 34%, while simultaneously reducing\nthe friction coefficient in extended sliding tests. The practical implication of the current findings is\nthat a combined\nCPB–LST approach could prove an excellent means of reducing\nfrictional response (by up to 99.4%) in non-conformal bearings functioning\nunder controlled abrasive wear conditions.",
"introduction": "1 Introduction Global energy demand is expected to see a 37% increase by 2040, 1 while fossil fuel emissions are forecast to outweigh\nsavings from renewables so that a catastrophic 2 °C rise in average\ntemperature will be hard to avoid. In the quest to combat climate\nchange, a high-impact measure is cutting the energy consumption of\n∼1.2 billion vehicles in service today. A prime candidate is\nthe estimated 57% of energy supplied to electric vehicles (EV) which\nis wasted on friction 2 , 3 and extenuated by the exponential\nincrease in the EV adoption rate. Also, in internal combustion engines\n(ICEs), friction losses still constitute 11.5% of the total fuel energy. 4 Reducing mechanical friction through improved\nsurfaces is thus one of the most effective ways to improve energy\nefficiency and reduce material waste. To this end, automobile and\nlubricant manufacturers have been implementing ways to reduce friction\nlosses. These range from start–stop systems to limiting the\nICE running time to more focused tribological approaches, such as\nthe adoption of low-viscosity oils, 5 polymeric\ne-lubricants (smart rheological), 6 laser-produced\nsurface texture coupled with mirror polishing of cylinder liners, 7 and polymer-coated journal bearings. 8 An effective way to facilitate sliding\nbetween components in contact\nis to densely anchor assemblies of polymer chains poly(methyl methacrylate)\n(PMMA) on the surface of solid materials. Recent advances in surface-initiated\ncontrolled radical polymerization allow the growth of uniform polymer\nbrushes, 9 − 11 while increasing the graft density by more than an\norder of magnitude compared to typical semi-dilute polymer brushes. 12 This type of surface enhancement is referred\nto as concentrated polymer brushes (CPBs). Researchers from Kyoto\nUniversity and Tsuruoka College have successfully shown that CPBs\ncan reduce friction by 2 orders of magnitude, constructing a robust\nlubrication system, which can achieve superlubricity (μ min < 10 –2 ) in both nanoscopic 10 − 12 and macroscopic tribological contacts. 13 In addition, these CPBs enable a 10-fold increase in the thickness\nof the brush layer compared to conventional semi-diluted polymer brushes.\nThis increase in thickness enhances the durability under sliding contacts 14 and could thus be applied to mechanical components,\nincluding journal bearings, sealing devices, and piston assemblies. Despite promising significant energy reductions, CPBs have escaped\npractical application because of two limitations: (i) lengthy exposure,\nhigh vacuum, and high temperature cause the rapid evaporation of most\nswelling agents such as organic solvents, leading to the loss of superlubricity\nand (ii) severe contact conditions reduce wear resistance capabilities.\nResearch by Tsujii and Sato 15 successfully\naddressed the former concern by employing an ionic liquid (IL) as\na swelling agent. ILs help preserve the swollen state of polymer brushes\nover long periods of time and under high vacuum or high-temperature\nconditions due to the liquid’s minimal volatility and flammability,\ncombined with relatively high thermal stability. 16 Furthermore, researchers recently achieved a robust system\nby grafting IL polymer brushes onto smooth surfaces, which resulted\nin very low friction (μ min ≈ 10 –3 ) for applied normal loads as high as 15 N. 13 The same order of magnitude of friction coefficient was recently\nrecorded by Tadokoro et al., 17 who employed\na custom-made apparatus to study the impact of well-swollen PMMA–CPBs\non friction, clearance, and leakage rate in reciprocating seals. Although recent polymer brush studies have shown exceptional frictional\nstability for up to 5000 cycles, 13 , 14 limited preservation\nof brushes under severe high-pressure high-temperature operating conditions\nstill prevents use on an industrial scale. To address this, the current\nstudy puts forward a combined friction and wear reducing surface modification\ntechnique, consisting of CPBs grafted onto surfaces initially laser-etched\nwith a matrix of micron-sized features—the smallest texture\ndimensions so far reported in the tribology literature. Applying\nlaser surface texture (LST)—that is , features\nsuch as pockets or grooves—to the surface of components\nis a way of improving lubrication that has been investigated since\nthe 1960s. 18 The impact of this approach\ncan be significant. In fact, it has been consistently shown to give\nfriction reductions of up to 80% in controlled laboratory tests. 19 − 28 Compared to other energy-saving solutions, LST is of low cost and\neasy to implement. It does not require components to be redesigned\nand can be incorporated into existing and new technologies. A recent\nseries of studies at Imperial College has elucidated the tribological\nmechanisms associated with surface texture and explained earlier discrepancies,\nby highlighting the critical dependency on contact conditions. Under\nboundary and mixed lubrication conditions (i.e. when the lubricant\nlayer between component surfaces is too thin to prevent solid–solid\ncontact), pockets consistently boost fluid film thickness (probably\nas a result of cavitation-driven flow 29 termed “inlet suction”) and thus drastically reduce\nfriction. 30 This is practically beneficial\nsince many lubricated automotive components (pistons, cams, and gears,\namong others), routinely operate under mixed lubrication conditions.\nSpecific pocket geometry criteria (shape, orientation, and spacing)\nhave also been show to further reduce friction. 20 , 27 When optimized in this way, surface texture coverage as low as 5%\ncan generate friction reductions of up to 82%, compared to nontextured\ncomponents. 21 Alternatively, macroscale\ntexture has recently been shown to reduce friction in the full-film\nregime, thanks to the shear area reduction mechanism. 31 In addition, surface texture has been shown to reduce wear\neither by increased surface separation due to film thickness increase\nand/or by acting as debris traps. 20 , 21 In a\nseries of recent studies, Watanabe et al. 32 , 33 combined IL swollen CPBs with micrometer-sized grooves and nano-periodic\nstructures to achieve a significant increase in durability compared\nto CPBs grafted onto non-textured surfaces. It was shown that while\ngrooves parallel to the direction of sliding act to increase the friction\ndurability of CPBs by up to 36%, 32 applying\nthese micro-grooves on nano-periodic-structured surfaces improve the\ndurability of CPBs by up to 90% compared with CPBs grafted onto non-textured\nsurfaces. 33 In a subsequent study,\nMiyazaki et al. 34 examined the durability\nof PMMA–CPBs applied on substrates\nwith chemically etched parallel grooves of different dimensions. Through\na combination of lubricated sliding and nanoindentation tests, the\nauthors proposed a durability enhancement mechanism created by the\n“layered structure” of the concentrated polymer brush.\nNanoindentation tests showed that when grafted onto non-textured surfaces,\nCPBs display a structure comprising two layers: a diluted “surface\nlayer” and a more concentrated “bulk layer”.\nWhen the CPBs were grafted onto a textured substrate, an additional,\nthird layer (i.e., a “reinforced tough layer”) forms\ninside the concave space of the parallel grooves, acting to enhance\nthe durability of the CPBs. The aim of the current study was\nto improve friction and wear efficiency\nby understanding and exploring the inter-dependencies between CPBs\nand laser-produced surface textures. To achieve this, a novel test\napparatus was designed and built at Saitama University to allow in\nsitu visualization of CPB-collapsing behavior and shed light on previously\nhypothesized wear mechanisms of CPBs. Tests were conducted under the\nmost extreme conditions that CPBs can endure (located on the static\nsurface of a sliding contact pair and thus subject to constant high\ncontact pressure throughout the test duration), with the aim of demonstrating\ntheir real-life potential to reduce energy losses and improve machine\nefficiency. This is underpinned by measurements to understand the\nmechanistic interactions between CPBs and LST and hence assess the\npotential of micron-size pockets to effectively reduce wear and consequently\nincrease the life of CPBs by trapping debris particles (as shown by\nthe authors recently for steel-on-steel reciprocating contacts 21 ). Based on this, a new anti-wear strategy is\nput forward, in which femtosecond laser-textured micro-cavities suppress\nthe propagation of CPB failure through the offered additional lateral\nsupport and thus increase the durability.",
"discussion": "4 Results and Discussion This section demonstrates the benefits of CPBs in terms of sliding\nfriction and their sliding distance/stress-related limitations. The\nimpact of geometrical surface-textured parameters on friction force\nand film thickness are also characterized as well as the ability of\nmicro-features to reduce CPB wear. Finally, results are summarized\nand a new friction and wear reduction mechanism for this combined\nCPB–LST is put forward. 4.1 Influence of CPBs on Friction\nForce and Their\nWear Behavior 4.1.1 Compression of the Diluted\nSurface Layer\nunder Static Conditions To investigate the time-dependent\ncompression of the CPB “diluted surface layer” (i.e.,\na layer with a low elastic modulus initially identified by Miyazaki\net al. in ref ( 34 ) following\na series of nanoindentation tests), Figure 4 shows the combined CPB-lubricant film thickness\nvariation recorded for three non-consecutive tests over a period of\n90 s. These repeat tests were performed using a non-textured sample\nunder static conditions (zero sliding speed) with an applied normal\nload of 5 N. In addition to the high measurement repeatability, this\nshows that the combined CPB-lubricant film (i.e., IL) gradually decreases\nover the 90 s period from a maximum swollen height of 715 nm to an\naverage height of 637 nm. The gradual film reduction suggest that\na squeeze flow process is more likely to be occurring than the rotation\nof the brushes. Figure 4 Film thickness decline over time for the CPB-coated, non-textured\nspecimen; repeatability between three different tests. Inset—schematic\nrepresentation of the time-dependent compression process of the diluted\nsurface layer for a constant applied load of 5 N. The pictogram highlights\nthe change between the initial position of the polymer brushes immediately\nafter the contact is loaded and their final orientation at the end\nof the static test. At the start of the test, the load is supported\nby a small number of adhesion points and the surrounding IL; this\nincreases gradually as the diluted surface layer is compressed and\nthe load becomes carried by a more homogeneous, concentrated bulk\nlayer. As the combined CPB-lubricant film decreases and the number\nof adhesion points grow, the diluted surface layer, crucial for the\nultralow friction of CPBs, 33 is gradually\ncompressed to become a concentrated bulk layer. Red dotted line shows\ntheoretical squeeze film thickness predicted by eq 3 . This can be analyzed by considering a viscous fluid between plates,\nunder zero sliding speed, which has a time-dependent film thickness\npredicted using Reynolds equation 1 where h liq (0)\nis the initial film thickness, W is the applied load, R is the radius of contact (estimated using Hertz theory\nto be ∼307 μm), and η is the viscosity. The film\nthickness, h liq , in eq 1 is that of the “liquid-like”\ndilute layer (consisting of polymer brushes in the IL solvent) as\nidentified by Miyazaki et al. in ref ( 34 ) since it is the one that will flow out of the\ncontact under load. Therefore, the total film thickness (which is\nmeasured and plotted in Figure 4 ) is given by 2 where h sol is\nthe constant thickness of the “solid-like” concentrated\nbulk layer (which does not flow out of the contact). Combining eqs 1 and 2 gives 3 Equation 3 can\nthen\nbe fitted to the data in Figure 4 (as shown by the yellow dotted line), with the only\nadjustable constants being the viscosity, η, and the concentrated\nbulk layer thickness, h sol . This yields\na viscosity value of ∼50 mPa s (which is similar to that of\nneat MEMP–TFSI 15 and a concentrated\nbrush layer thickness of ∼605 nm, which is consistent with\nindependent indentation measurements 34 ).\nThe close match between eq 3 and the measured film thickness, combined with the reasonableness\nof the resulting brush thickness and viscosity estimates, supports\nthe hypothesis that squeeze flow is responsible for the observed decay\nin film thickness. Note that the long duration of the process (occurring\nover 10 s of seconds) is a result of the CPB’s low stiffness\nand hence large contact radius, which requires time for the fluid\nto flow across. 4.1.2 Impact of the Sliding\nDistance and Surface\nTexture on the Wear Behavior of CPBs To assess the wear behavior,\na series of extended sliding tests (60 s each) was performed. The\nresulting frictional response of two textured patterns (Ø5 – d 0.2 and Ø10 – d 0.2)\nis compared against the non-textured, CPB-coated reference ( Figure 5 a). The combined\nCPB-IL film thickness is shown quantitatively ( Figure 5 b) as well as qualitatively ( Figure 5 a—details and Videos S2 , S3 , and S4 ). The textured patterns of identical depth\nand different diameter (5 and 10 μm) and also the smooth reference\nwere tested at the two lowest speeds of 0.2 and 0.6 mm/s. Figure 5 Simultaneous\nfriction force (a) and film thickness (b) comparisons\nbetween all CPB-coated, textured and non-textured disc samples, recorded\nover the 60 s wear tests (test conditions: sliding speed: 0.2 and\n0.6 mm/s; applied load: 5 N; lubricant temperature: 25 °C); Inset—interferometry\nimages captured for all three samples after 20 s of sliding (sliding\nspeed: 0.3 mm/s). (c) Shear stress versus strain rate; data replotted\nfrom Figure 5 a,b. The film thickness from the static case (i.e. , data from Figure 4 ) is also shown in Figure 5 b. Once the diluted surface layer of the CPBs is compressed/squeezed\nout and the combined CPB-lubricant is reduced to around ∼600\nnm (i.e. , the thickness of the concentrated bulk\nlayer), a close agreement is observed between the frictional response\nand the squeezing time of the more concentrated CPB layer ( Figure 5 ). Figure 5 a shows\nthat the Ø10 – d 0.2 textured pattern\ndisplays both the lowest initial friction response and the greatest\nwear durability at both sliding speeds. The non-textured reference\nhas the weakest performance and shows accelerated degradation after\n∼50 s, with the film thickness gradually reducing to around\n200 nm. The three images attached to Figure 5 a display the CPB film for all three samples,\ncaptured\nafter 20 s of sliding at 0.6 mm/s. While for both non-textured and\nØ5 – d 0.2, the CPB collapse has already\nstarted; no sign of wear is apparent for the Ø10 –\nd 0.2 textured pattern. However, as shown in Video S3 , after 20 s, and as highlighted in Figure 5 a, the CPB layer on the Ø10 – d 0.2 sample collapses entirely in less than 4 s.\nAlthough the CPBs on the Ø5 – d 0.2 sample\nare the first to show signs of degradation and the narrower micro-features\ndelay the collapse, increasing the life of the polymer brushes by\n∼10 s (5.5 mm). This behavior can also be observed in Figure 5 b, which shows a\nprolonged resistance of the combined CPB-IL film thickness for the\nØ5 – d 0 . 2 sample when\nsliding at 0.6 mm/s. For all textured surfaces, the collapse\nof the CPBs always commenced\nalong the dimple-free zone at the center of the contact, where two\nconsecutive lines of pockets were omitted during laser micromachining.\nWe hypothesized in ref ( 34 ) that this wear reduction behavior due to surface texture, which\ncan be seen in all corresponding videos, is generated by the presence\nof a third, “reinforced tough layer”, located inside\nthe concave space of the textured micro-features, where the polymer\nchains are grafted onto both the bottom of the pockets and the “vertical”\nsidewall surfaces. This enhanced CPB durability that delays/decelerates\nwear is supported by the visual proof presented in Figure 5 and Videos S2 – S4 (see whole videos in\nthe Supporting Information). Figure 5 a,b shows\nthat, for each sample, friction increases as film thickness (i.e. , shear rate) decreases. This may be due to the viscous\nnature of the film within the contact and can be investigated as follows.\nThe friction data in Figure 5 a can be converted into shear stress, τ, by dividing\nby the contact area ( A = π R 2 = π × (307 × 10 –6 ) 2 = 2.96 × 10 –7 m 2 ), and\nthe measured film thickness in Figure 5 b can be converted to strain rate by γ̇\n= Δ u / h liq = Δ u /( – h sol ), where h sol is the concentrated\nbrush\nlayer thickness, which was estimated from Figure 4 to be ∼605 nm. The resulting plot\nof shear stress against the strain rate in Figure 5 c shows a linear relationship between shear\nstress and strain rate suggesting Newtonian fluid behavior (τ\n= ηγ̇). There is variation between the slopes of\nthese lines, which may be due to deviations in the polymer brush thickness\n(not always 605 nm as assumed). However, the average gradient (i.e. , a prediction of the viscosity) is 58 mPa s with a standard\ndeviation of 20 mPa s. This is remarkably close to the 50 mPa s predicted\nby Figure 4 and confirmed\nby the literature 15 and strongly suggests\nthat the observed friction behavior is dominated by the viscous film\nbehavior of the MEMP–TFSI solvent. It should also be noted,\nhowever, that the time until failure of the CPB-coated specimens in Figure 5 a,b (denoted by an\nabrupt reduction in film thickness) is closely linked to the sliding\ndistance rather than time, for both the 0.3 mm/s and the 0.6 mm/s\nsliding speeds tests; the total sliding distance to collapse was approximately\n16 mm (i.e. , 2× the sliding speed took 1/2 the\ntime to fail). 4.1.3 Ability of CPBs to Reduce\nFriction and Possible\nCauses of Wear Throughout this study, CPBs were tested under\nsevere contact conditions, being at all times located on the stationary\ncomponent of the tribo-pair. The same polymer brushes located inside\nthe contact on initial loading were thus subjected to friction and\nwear throughout the entire test. Previous studies describing the lubrication\nmechanism of CPBs were performed under milder conditions, with polymer\nbrushes generally located on the moving surface. In the latter situation,\nthe contact is continuously replenished with fresh, fully swollen\nbrushes. Moreover, the non-conformal point contact investigated in\nthis study led to a high contact pressure of 25 MPa being consistently\napplied on the polymer brushes. It is not possible to plot standard\nStribeck curves (friction vs speed) for the CPB-grafted samples in\nthis study, since the coefficient of friction is not only a function\nof sliding speed but also of sliding distance and duration (as shown\nabove). Hence, to assess the ability of CPBs to provide low friction, Figure 6 plots two sets of\ndata, each obtained following three repetitions: (i) the frictional\nresponse recorded for the non-CPB coated, non-textured reference,\nobtained over a range of sliding speeds varying between 0.2 and 2000\nmm/s (i.e. , classic Stribeck curves), and (ii) a\nlimited frictional data set for the CPB coated, non-textured sample,\nconsisting of the coefficient of friction during the initial step\n(sliding speed: 0.2 mm/s) and the frictional response at 60 mm/s,\nwhere the CPB film is instantly collapsed by the shear stress breaking\nthe anchoring bonds between the brushes and the surface. Figure 6 Stribeck curves\nshowing the friction behavior for the non-CPB coated\nreference, plotted alongside the coefficient of friction recorded\nat 0.2 and 60 mm/s for the CPB-coated sample (picture insets showing\ninterferometry images captured for the CPB-coated sample immediately\nafter sliding movement begun and when CPB collapse. Graphical insets\nshow friction force variation as recorded during the 0.2 mm/s step\nmeasurement—the red square representing the time interval where\nthe friction was averaged. Applied load was kept constant at 5 N,\nwhile sliding speed was varied from 0.2 to 2000 mm/s. As expected, the non-CPB coated sample produces a standard\nStribeck\ncurve, showing the transitions between boundary, mixed, and hydrodynamic\nlubrication regimes. For the non-textured, non-CPB-coated sample,\nthe highest friction is recorded at low speeds, as insufficient lubricant\nis entrained between the surfaces and load is supported by asperity\ncontact. There is then a steep decrease in friction as speed increases,\nand more lubricant is entrained to separate the surfaces with an easily\nsheared IL layer (i.e. , mixed lubrication regime).\nFinally, as the sliding speed increases above 200 mm/s, the bearing\nshifts to the full film regime and friction rises again because of\nincreased shearing of IL layers. When comparing the CPB-coated\nsample with the non-coated reference,\nsignificant reductions in friction of up to 99.4% were observed during\nthe initial sliding step (representing the difference in average friction\nof the data points recorded when the sliding speed was set at 0.2\nmm/s, highlighted by the graphical insets shown in Figure 6 ). The low-speed friction of\nthe CPB specimen is less than 0.01 and is sufficiently low to be classed\nas superlubricity. Figure 6 also shows\nthat the benefits from CPB grafting vanish at sliding speeds above\n60 mm/s, which corresponds to the transition from mixed to a full\nfilm regime. To further understand the causes of this reduction in\nfriction performance, the contact was viewed using the interferometry\nset-up. As shown in Figure 6 and Video S1 , the CPBs were entirely\nremoved from the surface in less than 1 s from the start of each test.\nIt should be noted here that a fresh coated specimen was used for\neach measurement point. After inspecting Figures 5 and 6 , the following\ncomments can\nbe made about the failure of CPBs under submerged sliding conditions:\n(i) time to failure is proportional to the sliding distance ( Figure 5 ) in agreement with\nan Archard coefficient type law, (ii) the IL is gradually squeezed\nout of the sliding contact (the higher the speed, the faster this\nhappens), (iii) the shear stress (proportion to friction coefficient)\nincreases as the film thickness decreases and may reach a level sufficient\nto cause scission of the brushes themselves or scission of the bonds\nbetween brushes and substrate, and (iv) localized failure occurs and\nleads to a stress concentration that causes other regions to follow\na scraping type of wear. 4.1.4 CPB Layered Structure\nBehavior with Increasing\nSliding Speed The combined CPB-IL film thickness was measured\nfor all tests on CPB-coated samples. Figure 7 shows one example for a test carried out\nwith the Ø5 – d 0.2-textured sample. As\nsliding speed increases, the CPBs are compressed. To avoid situations\nwhere the CPBs are removed completely, leading to direct contact between\nthe disc’s chromium layer and the steel ball (as occurred in Figure 6 ), the test duration\nwas adjusted accordingly for each sliding speed step. In each of these\ntests, the first 0.2 s represent the loading step. This initial, static\nperiod can be identified in the film thickness chart for the 6 mm/s\nspeed case ( Figure 7 ), where the recorded values show an accelerated collapse of the\nCPBs as soon as sliding motion starts. Figure 7 Combined CPB-IL film\nthickness captured for the Ø5 – d 0.2\ndisc specimen (sliding speed: 0.2, 0.6, 2, 6, and 20\nmm/s, applied load: 5 N; lubricant temperature: 25 °C). Inset:\ninterferences fringes obtained initially and after 3 s. Although not shown in Figure 7 , the contact was unloaded after each speed measurement\nfor a period of 60 s. This highlights the behavior of the CPB-“layered\nstructure”, introduced in ref ( 34 ) and discussed above. It is likely that in this\nexample, the “diluted surface layer” is approximately\n50 nm, reduces in thickness during the first two speed measurements\n(0.2 and 0.6 mm/s), and more rapidly during the third speed measurements\n(2 mm/s). During this latter step, film reduction gradient changes\n(at ∼800 nm), probably indicating that the solid-like “bulk\nlayer” is beginning to support the applied load. A similar\nbehavior is observed at 6 mm/s. Figure 7 shows the almost complete recovery of the “diluted\nsurface layer” between the first three speed steps (indicated\nby the arrows), followed by a partial recovery between the third (2\nmm/s) and fourth (6 mm/s) speed steps. However, following the fourth\nstep, no recovery occurs which suggests permanent deformation of this\ntop layer occurred at a combined CPB-lubricant film thickness of ∼700\nnm. At 20 mm/s, the film decays more rapidly due to increased sliding\ndistance. 4.2 Parametric Summary of LST–CPB\nFriction Figure 8 show friction\nversus sliding speed and distance performance of all four textured\nsamples (with varying dimple depth and diameter) and the non-textured\nreference. For all specimens, friction force increases with sliding\nspeed and distance due to decreasing film thickness. Initially, the\nnon-textured specimen shows highest friction possibly due to its larger\ncontact area. Then, as sliding progresses, the specimens with deepest\npockets show highest friction probably due to increased squeeze flow\nof IL out of the contact leading to a thinner lubricant film. However,\nthe shallower Ø5 – d 0.2 pocketed consistently\nshows lower friction probably due to enhanced anchoring of the polymer\nbrushes combined with minimum squeeze flow. Figure 8 Friction comparison between\nall CPB-coated textured samples and\nthe non-textured reference. The observed improvements for textured specimens are attributed\nto the lateral support offered by the polymer brushes situated inside\nthe textured features (i.e. , the “reinforced\ntough layer”) to the brushes grafted outside the pockets (i.e. , the “concentrated bulk layer”), thus delaying\nthe decline in film thickness. Naturally, polymer brushes situated\ninside deeper pockets offer less support and lead to greater reduction\nof the CPBs on the sample’s surface. At the lowest sliding\nspeed of 0.2 mm/s, the textured pattern Ø10 – d 0.2 displayed the lowest frictional response, down to a\ncoefficient of friction of just 0.0006. This improved performance\nof Ø10 – d 0.2 is attributed to an increased\nmaterial pileup around the edges of the pockets (i.e. , 200 nm tall spikes, generated by the laser texturing process— Figure 8 , Legend). The laser\nparameters were deliberately modified to achieve these spike features\nin order to alleviate CPB wear compared to regular pockets. When polymer\nbrushes were grafted onto this textured surface, the pileup spikes\noffered additional lateral support to the “concentrated bulk\nlayer” and the “reinforced tough layer” of the\nCPB structure, reducing their compression and thus boosting their\nfriction performance. 4.3 Impact of Abrasive Particles\non the CPB Film The three chromatic LED images in Figure 9 a (and Videos S5 in Supporting Information) show how\na wear debris particle travels\nalong the contact, damaging the CPB film through abrasion, leading\nto its collapse. Contrastingly, the halogen light image in Figure 9 b shows a textured\ncontact in which CPB wear initiates and gradually progresses along\nthe dimple-free zone. However, beneficially, wear particles which\nenter the contact along the textured area do not cause any damage\nto the CPB coating. This may be due, as we have recently suggested, 34 to surface texture providing an additional third\nlayer inside the concave space of the pockets that increases wear\nresistance along the textured region by suppressing the propagation\nof CPB failure by offering additional lateral support to the surface\nand bulk layers. Figure 9 (a) Wear debris particle passing through a CPB-coated,\nnon-textured\ncontact and the subsequent wear damage of the CPB layer; (b) successive\npositions of a CPB-coated, laser-textured contact (Ø5 – d 0.2) showing: accelerated wear along the dimple-free area,\nreduced damage due to wear debris passing through the textured area,\nand the collapse of the combined CPB-IL film. Considering the CPB wear behavior illustrated in Videos S5 and S6 , it is suggested\nthat although CPB–LST combinations significantly reduce friction\nforce (by up to 99.4%), this should currently only be considered in\ncontrolled wear environments free from abrasive conditions, such as\nstatic sealing, near-vacuum environments, or space tribology applications."
} | 8,331 |
26019230 | PMC4512042 | pmc | 6,265 | {
"abstract": "Plants live in a social environment, interacting with the roots and shoots of neighbours. Life with neighbours is a chronic stress, with different behaviours altering the social dynamics. To understand root behavioural strategies in response to neighbours, we observed root growth for 20 species. There was no single response, and instead a continuum of responses from avoidance to aggregation near neighbours. Species were capable of two strategies, (1) location-sensitivity, adjusting the vertical and horizontal placement of roots, and (2) size-sensitivity, reducing root system size. Overall, there is surprising complexity in how plants respond to social environments, with implications for resource use, coexistence, and production.",
"conclusion": "Conclusions Here we used a comparative approach to identify novel behavioural strategies in how plants alter root growth in response to neighbours. Our findings highlight the need to consider species identity when predicting response to neighbours, rather than expect a single dominant strategy of over-proliferation, avoidance or neutrality. Instead, all of these behavioural responses were observed among different species. Though such idiosyncratic responses increase the difficulty of understanding, they do indicate it is critical to understand the biology of the specific species involved in any social interaction. We confirmed prior findings that some species have the potential to alter their fine-scale horizontal and vertical root placement behaviour in response to neighbours, even without showing a negative growth consequence of the ‘competitor’. This potentially has important implications for species coexistence, and may be a behavioural trait-filter influencing community assembly and ecosystem function.",
"introduction": "Introduction The close proximity of neighbours, combined with strongly overlapping resource requirements, results in competition for limiting resources being a commonly experienced ecological interaction among plants. Competition can greatly reduce individual fitness and alter evolutionary trajectories ( Keddy 2001 ). At the community level, competitive interactions can lead to competitive exclusion, may alter community structure among co-occurring species ( Lamb et al. 2009 ) and can influence plant invasions ( Levine 2001 ; Gurevitch et al. 2011 , Bennett et al. 2014 ). Thus, competition has the potential to alter fundamental aspects influencing the evolution, persistence and coexistence of species in natural and managed landscapes. Despite the importance of competition at many organizational scales, and despite it being an inherently social interaction, only recently have ecologists explicitly focused on understanding plant competition through a behavioural lens (e.g. Gersani et al. 2001 ; Cahill and McNickle 2011 ; McNickle and Brown 2014 ). Here, we build upon behavioural concepts and approaches to better understand how plants alter root growth in the context of social interactions. In many systems, particularly herbaceous communities such as grasslands, the majority of plant biomass is belowground ( Schenk and Jackson 2002 ). Additionally, when measured, root competition is often a more severe limitation to plant growth than is competition aboveground ( Casper and Jackson 1997 ). Nonetheless, our understanding of plant responses to neighbouring shoots is substantially more advanced (e.g. Smith and Whitelam 1997 ) than our understanding of plant responses to neighbouring roots ( Cahill and McNickle 2011 ). Better information of how plants alter growth patterns and modify patterns of soil occupancy in response to neighbouring roots should advance our understanding of the causes and consequences of competition and coexistence. By using concepts drawn from the field of behaviour, what a plant does in response to some change in the biotic or abiotic environment ( Silvertown and Gordon 1989 ), one can draw upon a rich conceptual foundation to understand deterministic and plastic growth patterns in plants. There is substantial evidence that many species of plants have the capacity to alter patterns of root placement in response to neighbours ( Schenk 2006 ; reviewed in Cahill and McNickle 2011 ). The general patterns found include spatial segregation of neighbouring root systems ( Baldwin and Tinker 1972 ; Brisson and Reynolds 1994 ; Caldwell et al . 1996 ; reviewed in Schenk et al . 1999 ; Holzapfel and Alpert 2003 ), over-proliferation of roots in the area of potential interaction ( Gersani et al . 2001 ; Maina et al . 2002 ; Padilla et al. 2013 ), along with examples of no response ( Litav and Harper 1967 ; Semchenko et al . 2007 ). Behavioural responses to neighbours appear species specific, and can change as a function of neighbour identity ( Mahall and Callaway 1991 ; Falik et al . 2003 ; Bartelheimer et al . 2006 ; Fang et al . 2013 ). Despite the strong evidence that plants exhibit complexity and contingency in how they occupy and explore the soil environment ( Mommer et al. 2012 ), the research performed to date is predominately a series of individual studies with idiosyncratic methods and measures, species selections and variable results. Lacking has been a broadly comparative approach to understanding how plants respond to the roots of neighbours ( McNickle and Brown 2014 ), analogous to efforts to understand how plant roots respond to the spatial distribution of soil nutrients ( Campbell et al . 1991 ). How a plant modifies its occupation of the soil environment in response to a neighbour has important implications for competition for limiting soil resources. Root segregation could result in habitat differentiation, leading to a lack of a ‘shared’ resource pool, and thus enhancing coexistence ( Silvertown 2004 ). In contrast, plants which tend to aggregate roots at the zone of interaction may exaggerate the spatial overlap of soil depletion zones, leading to enhanced competitive interactions ( Gersani et al . 2001 ). Though there is no existing theory describing which kinds of species are more or less likely to be segregators, aggregators or non-responders in the context of root interactions, there is a theory available in the context of how plants alter root placement and foraging behaviour in response to patchily distributed soil resources. Campbell et al . (1991) predicted that ‘large scale foragers’ (plants with large root systems) will exhibit little ability to precisely place roots in nutrient patches, while smaller scale foragers will have greater ability to finely adjust root distribution. A phylogenetically controlled meta-analysis did not find support for this prediction ( Kembel and Cahill 2005 ). Instead, Kembel and colleagues ( 2005 , 2008 ) found that foraging precision in relation to nutrients was positively associated with a number of traits typically associated with weediness and ruderal life-history strategies. How size, competitiveness and other plant traits are associated with plant responsiveness to neighbours is unknown. In this study we experimentally test three specific questions. (i) Are there general patterns in an individual's root behaviour to neighbouring plants among 20 co-occurring grassland species? (ii) Is a plant's root behaviour contingent upon neighbour identity? (iii) What other plants traits are associated with root behavioural strategies? To answer these questions, we visualized roots using a window box apparatus, allowing for root identification and quantification.",
"discussion": "Discussion General patterns In previous work, root behavioural responses to neighbours have varied from no response (e.g. Litav and Harper 1967 ; Semchenko et al. 2007 ), to segregation (e.g. Baldwin and Tinker 1972 ; Brisson and Reynolds 1994 ; Caldwell et al . 1996 ; Schenk et al . 1999 ) or over-proliferation (e.g. Gersani et al . 2001 ; Maina et al . 2002 ; Padilla et al. 2013 ). Results presented here (Fig. 1 ) are consistent with the lack of consistency in these prior findings. We suggest that such behavioural variation is now well demonstrated, and we argue against a strict interpretation of the ‘Tragedy of the Commons’ prediction of over-proliferation of roots in the zone of competitive encounters ( Gersani et al . 2001 ). Instead, the variation in behaviour observed here, and in prior studies, is consistent with a broader view that multiple adaptive strategies may occur when plants play competitive games ( McNickle and Dybzinski 2013 ; McNickle and Brown 2014 ). We also note that neither observing behavioural variation in root responses to neighbours, nor modelling fitness differentials associated with different behavioural types is equivalent to demonstrating these behaviours are adaptive. Again, the study of plant foraging behaviour is substantially behind the understanding of the adaptive value of competitive behaviours aboveground, such as the shade-avoidance response ( Dudley and Schmitt 1996 ). We suggest that more focus on testing the fitness consequences of alternative foraging behaviours is a potentially fruitful area for future research. Though there was a substantial variation in how plants responded to neighbours, we found no evidence that responses were functionally different among monocots and eudicots. This was surprising, as Kembel and Cahill (2005) found broad differences in the root foraging plasticity of monocot and eudicot species in response to nutrient heterogeneity. Furthermore, both Cahill et al. (2008) and Kiær et al. (2013) showed different competitive effects among monocots and eudicots, and thus we had expected to see clustering of these two groups in terms of behaviour in response to neighbours. We are unable to determine whether our lack of response was due to our relatively limited phylogenetic representation (only 20 species), or whether our results indicate a lack of phylogenetic bias in the tendency to alter root behaviour in response to neighbours. Similarly, we also found no consistent effect of neighbour identity on root responsiveness to a neighbour. Although previous studies have not always included neighbour identity as a variable for investigation, when they have the comparison is usually between inter- and intra-specific competition ( Mahall and Callaway 1991 ; Bartelheimer et al. 2006 ) or genotypes of the same species ( Callaway and Mahall 2007 ; Dudley and File 2007 ; Murphy and Dudley 2009 ; Fang et al. 2013 ). Evidence suggests that some plants are able to identify their neighbours at the root level ( Chen et al. 2012 ), and that some species can alter their root responses according to that identity ( Mahall and Callaway 1991 ; Bartelheimer et al. 2006 ; Callaway and Mahall 2007 ; Dudley and File 2007 ; Murphy and Dudley 2009 ; Fang et al. 2013 ). It is unclear why we found no similar effect here; though caution that it is difficult to draw strong conclusions, as only two neighbour species were used. Species-level responses and root behavioural strategies As mentioned previously, we chose these 20 focal species to be representative of the species that co-occur in a local grassland; they were not chosen to test species-specific hypotheses regarding behavioural responses and strategies. Consequently, each species received relatively little replication, with the strength of the data coming from the comparisons among species. Though these data can be used to test a number of ecologically relevant questions [e.g. are specific root behavioural types associated with high/low abundance in natural system; do specific behavioural types influence a species' response to other ecological challenges (e.g. herbivory)], such questions are well beyond the scope of this manuscript. Instead, we limit our discussion to the two novel behavioural strategies we have identified which are used by plants in response to growing with a neighbour (Fig. 4 ): size-sensitivity and location-sensitivity. Size-sensitivity Nearly 40 % of the variations in species' root responses to neighbours were driven by changes in three size-related traits (total root length, change in maximum root system breadth and change in total biomass; Fig. 4 ). Not surprisingly, these were all positively correlated and indicate an overall reduction in plant size in response to growth with neighbours (i.e. net effects of competition). It is important to recognize, however, that associated with this reduction in plant size is also a reduction in the area of soil occupied by an individual's root system. Depending upon the allometry of these changes within an individual at the community level, there could be important implications for plant neighbourhood size, biomass distributions in the soil, the degree to which pools of limiting resources are shared among neighbours, as well as resource and host availability for mutualists and other members of the soil community. We suggest that this perspective on the ecological importance of shifts in soil occupancy patterns due to social interactions is overlooked within plant ecology, though widely recognized in the context of animal territoriality, density and resource availability ( Hixon 1980 ). Location sensitivity Not all focal species became smaller in response to growth with neighbours, such that there was no main effect of the presence or absence of neighbours for any response variable, including biomass measures [see Supporting Information— Table S1 ] . However, a lack of biomass effect does not equate to a lack of response to neighbours (Fig. 4 ). We found nearly 30 % of the variations in root responses to neighbours were associated with changes in biomass allocation (R : S ratio) and fine-scale changes in root placement (horizontal asymmetry and depth of maximum root system breadth), rather overall size. These changes indicate a second root system strategy incorporating behavioural plasticity, rather than simply gross biomass responses. We suggest that this is a potentially critical finding, as it highlights that the impacts of neighbours extend further than the traditionally studied resource limitation-biomass reduction paradigm. These data highlight a potential need to begin more robust exploration of the ‘non-resource’ consequences of neighbours on plant growth and coexistence, analogous to the rapidly increasing research into the non-consumptive effects of predators on prey populations (e.g. Peckarsky et al. 2008 ). The ability of plants to modify the fine-scale vertical and horizontal placement of roots in response to neighbours is well established (e.g. Mahall and Callaway 1991 ; Cahill et al. 2010 ; Mommer et al. 2010 ), and has a number of consequences for coexistence, invasion and ecosystem processes. Segregation of the roots of neighbouring plants has long been argued to be a mechanism allowing for species coexistence ( Parrish and Bazzaz 1976 ; Berendse 1983 , Craine et al. 2005 ), due to a reduction in the intensity of competition. The findings here suggest that such a differentiation in micro-scale habitat need not to occur only due to fixed traits of plants (e.g. deep- versus shallow-rooted species), but that behavioural modifications in response to local conditions are not uncommon among plant species. We suggest that reliance on fixed plant traits as a means of understanding the functional ecology of plants can lead to a significant misunderstanding of the mechanisms by which plants can interact with other plants and their environment. We suggest that location-sensitivity behaviours are a potential mechanism that could lead to enhanced coexistence and altered ecosystem functions, even in the face of a strong competitor. It may also be one potential mechanism by which plants are able to tolerate (in a fitness context), growing with aggressive neighbours. We found no support for the idea that our measures of root responsiveness were related to either plant size ( sensu the scale and precision ideas of Campbell et al. 1991 ), nor were they associated with the competition experienced by the focal plants. However, we believe that more work focussed on these root responsive strategies is needed, particularly in the context of fitness consequences, competitive tolerance and avoidance, community assembly and ecosystem function. We also agree with McNickle and Brown (2014) who suggest the accumulation of more and of different types of root trait data allows for novel insights into how plants forage and interact in the soil environment. We note several limitations in our identification of root responsiveness strategies, including a relatively small number of species (though more than have been used before), nearly two-dimensional growing conditions, short duration of the experiment, use of seedlings rather than mature plants and limited replication within species. How these strategies relate to fitness, the ability to perform in the presence of other ecological processes and non-foraging plant traits is also not known."
} | 4,260 |
32120900 | PMC7085567 | pmc | 6,267 | {
"abstract": "Wireless sensors are limited by node costs, communication efficiency, and energy consumption when wireless sensors are deployed on a large scale. The use of submodular optimization can reduce the deployment cost. This paper proposes a sensor deployment method based on the Improved Heuristic Ant Colony Algorithm-Chaos Optimization of Padded Sensor Placements at Informative and cost-Effective Locations (IHACA-COpSPIEL) algorithm and a routing protocol based on an improved Biogeography-Based Optimization (BBO) algorithm. First, a mathematical model with submodularity is established. Second, the IHACA is combined with pSPIEL-based on chaos optimization to determine the shortest path. Finally, the selected sensors are used in the biogeography of the improved BBO routing protocols to transmit data. The experimental results show that the IHACA-COpSPIEL algorithm can go beyond the local optimal solutions, and the communication cost of IHACA-COpSPIEL is 38.42%, 24.19% and 8.31%, respectively, lower than that of the greedy algorithm, the pSPIEL algorithm and the IHACA algorithm. It uses fewer sensors and has a longer life cycle. Compared with the LEACH protocol, the routing protocol based on the improved BBO extends the life cycle by 30.74% and has lower energy consumption.",
"conclusion": "5. Conclusions In order to reduce costs and save energy, this paper proposes a large-scale sensor deployment method called the IHACA-COpSPIEL algorithm and a routing protocol based on the BBO algorithm. Mutual information is introduced to describe the correlation between observed points and unobserved points, a mathematical model with submodularity is established, and the edges of graph theory are used to represent communication costs. The pSPIEL algorithm with enhanced optimization ability by a chaos operator and the ant colony algorithm with improved heuristic function and pheromone update mechanism are used to find the optimal path. What has been studied can further solve the sensor deployment problem under the constraint of communication cost. Finally, the BBO algorithm-based routing protocol transmits data to the deployed sensors. The computational complexity of the IHACA-COpSPIEL is O ( k N 2 ) , and the computational complexity of the routing protocol based on the BBO algorithm is O ( q n 2 ) . The experiments show that the deployment algorithm proposed in this paper has better sensor deployment capabilities. This deployment algorithm reduces the communication cost by 38.42% compared with the greedy algorithm. It also reduces the number of sensors and has a longer life cycle. Compared with the LEACH protocol, the BBO algorithm-based routing protocol has lower energy consumption and longer network life. In the future, we intend to use a discrete event simulator (DES) such as NS-3 to further combine practical application scenarios to improve the effectiveness of the algorithm. Our vision for future work is as follows. We will complete the IHACA-COpSPIEL protocol design in the NS-3. We will refer to the RFC document of Multi-Protocol Label Switching protocol, and elaborate on the design and implementation of each basic component of IHACA-COpSPIEL, including the forwarding equivalence class (FEC), next hop label forwarding entry (NHLFE), FEC to NHLFE mapping (FTN), etc. By statically configuring the label forwarding table, the communication between private networks through the backbone network by IHACA-COpSPIEL forwarding will be realized.",
"introduction": "1. Introduction Wireless sensors are widely deployed on a large scale in commercial fields [ 1 , 2 ], but are limited by node costs, communication efficiency between nodes, and energy consumption [ 3 , 4 , 5 ], e.g., in forest and grassland fire risk monitoring and early warning. The problem of wireless sensor deployment is considered as deploying a certain number of nodes to meet monitoring needs, that is, finding the number and location of deployed nodes. The goal of solving this problem is to find as few sensors as possible to meet the monitoring requirements and reduce the communication cost. It is transformed into an optimal sensor node solution set, which is an NP-hard problem. The sensor deployment problem has diminishing returns, e.g., submodularity [ 6 , 7 , 8 ]. Initially, when a small number of sensors are deployed, each new sensor will significantly improve its deployment utility. As more sensors are placed, the improvement in utility from adding new sensors diminishes. Krause [ 9 ] showed that for problems with submodularity, at least the ( 1 − 1 / e ) approximation of the optimal solution can be obtained using the greedy algorithm. Many methods have been proposed for sensor deployment. In [ 10 ], Huang et al. assumed that the node’s perception ability is a circular area. That is, targets within the circular area are fully perceived, and targets outside the circular area will not be perceived. In [ 11 ], Guestrin et al. proposed the use of mutual information-based optimization criteria so that the set of deployed nodes contains information about unselected points, and the monitoring is quite accurate. In [ 12 ], Cheng et al. proposed a Markov random field model to describe the data correlation between sensor nodes. In [ 9 ], Krause deployed sensors with a greedy algorithm to maximize the amount of information, but neglecting the influence of the communication distance between nodes. In [ 13 ], Krause et al. improved the greedy algorithm and proposed the Padded Sensor Placements at Informative and cost-Effective Locations (pSPIEL) algorithm to solve the problem of sensor deployment optimization under the constraint of communication distance; however, a large number of sensors needed to be deployed. In [ 14 ], Mariohat et al. established a Gaussian model, improved the greedy algorithm under the constraint of fixed costs, and proposed the SUPSUB method to minimize the submodular set function, while neglecting the influence of the communication distance between nodes on the deployment. The sensor placement problem considering communication distance is a constrained optimization problem. The bi-projection neural network proposed by Xia et al. [ 15 ] can effectively solve large-scale constrained optimization problems, and has good stability and faster convergence [ 16 ]. Liu et al. [ 17 ] proposed a ML-OAXSMT-PSO construction algorithm, which can significantly reduce the total cost. During communication transmission, wireless sensors have limited energy, but effective clustering nodes can better save energy and extend the life cycle of the entire network. There are various energy-saving methods. Guo et al. [ 18 ] proposed the FTAOA algorithm to minimize task completion time to save node energy. Cheng et al. [ 12 ] proposed the NSA algorithm to reasonably deploy nodes and significantly improve network lifetime. Liu et al. [ 19 ] proposed the KPNS algorithm to appropriately select more active nodes for monitoring, so that the energy is fully utilized. Effective node clustering can greatly save energy and extend the life cycle of the entire network. The LEACH protocol balances the energy of each sensor in the entire network by randomly selecting cluster heads [ 20 ]. However, it has the problem of uneven number and distribution of cluster heads [ 21 ], for not having considered the transmission distance. This causes either the nodes far away from the base station to be selected as the cluster head or the nodes far away from the cluster head to die prematurely [ 22 ]. In [ 23 ], Simon proposed the biogeography-based optimization algorithm with advantages of simple operation, few parameters, and high search accuracy [ 24 ]. In [ 25 ], Pal and others used the Biogeography-Based Optimization (BBO) algorithms to select cluster heads and cluster nodes, and obtained good energy efficiency. However, the authors only took the distance between cluster heads and the distance between nodes in the cluster into consideration, while neglecting the energy consumed by data transmission between nodes. Deploying wireless sensors is limited by cost and power consumption [ 26 ]. Therefore, the following two issues need to be considered during deployment: one is to achieve efficient data collection; the other is to use as few sensors as possible and minimize the communication distance between sensors to reduce the total energy consumption. Because of the existing problem of deploying fewer sensors in terms of the distance between nodes, and the deficiency of some popular algorithms in the field of sensor deployment, we shall proceed as follows in this paper. The mutual information is used to describe the correlation between the observed and the unobserved points. The communication distance is described by the connection of the graph and the Improved Heuristic Ant Colony Algorithm-Chaos Optimization of Padded Sensor Placements at Informative and cost-Effective Locations (IHACA-COpSPIEL) algorithm is used to choose the optimized submodular model. By considering the distance between clusters, the distance between nodes in the cluster, and the energy consumption of data transmission by the nodes, we obtain an optimized routing protocol in which the BBO algorithm is used to transmit data with an improved cost performance. The structure of this article is as follows. In Section 2 , wireless sensor deployment optimization is introduced. The routing protocol of the wireless sensor network based on the BBO algorithm is presented in Section 3 . In Section 4 , we introduce the experimental verification, discuss the deployment effects using different algorithms, and analyze the performance of the protocol for the BBO algorithm. Finally, this article is summarized in Section 5 ."
} | 2,434 |
37595873 | PMC10570954 | pmc | 6,271 | {
"abstract": "Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens . The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.",
"conclusion": "Conclusion The paradigm of the final steps of EET in G. sulfurreducens has been reassessed several times in the past 2 decades. The first models predicted that electrons reached extracellular acceptors through cytochromes embedded in the outer membrane facing the extracellular space or via pili-based nanowires ( 57 ). These models were revisited upon the discovery of cytochrome nanowires ( 10 ), which together with porin-cytochrome complexes are believed to be the main protagonists of the final steps of EET ( 9 ). Concomitantly, it has been suggested that Geobacter pili have a secretion role ( 58 ). Nevertheless, the pili-nanowire paradigm is still under debate ( 59 , 60 ). Using a combination of bioinformatic, structural, and functional studies, we found sufficient evidence that microbial heme-tethered redox strings provide an additional mechanism for the reduction of insoluble metal oxides in G. sulfurreducens and other electroactive bacteria ( Fig. 5 ). PgcA and homologs possess a tethered structure, in which different cytochrome domains are connected by low-complexity regions that participate in metal binding and warrant sufficient flexibility to promote intra- and intermolecular electron transfer at variable nanometer distances. Our results show that the cytochrome domains of PgcA from G. sulfurreducens can exchange electrons promiscuously through the formation of transient complexes, thus suggesting a cooperative mechanism of reduction of electron acceptors. In fact, while full-length PgcA has been shown to efficiently reduce Fe(III) oxides in vitro ( 18 ), our results revealed that such reduction is carried out by a single domain (Domain 3). Upon reduction, metal oxides detach from the protein and PgcA is available to be recharged with electrons. Depending on its cellular location, PgcA can have several putative redox partners. In the periplasm, the most likely candidates are either the inner membrane quinone oxidoreductases or the PpcA-family cytochromes ( Fig. 5 ). Consequently, during secretion, PgcA can be loaded with electrons and ready to reduce extracellular metals. Alternatively, if PgcA is secreted in an oxidized or intermediate redox state, it might work synergistically with cytochrome nanowires (OmcE, OmcS, and OmcZ) in the extracellular matrix, thus establishing a continuous flow of electrons for metal reduction. The overall efficiency of the process can be dependent on PgcA’s diffusion rates, as a consequence of differentiated protein-protein and protein-metal contacts, as well as biofilm density. Figure 5 Proposed model of extracellular electron transfer in G. sulfurreducens . The main protagonists of the electron transfer pathways towards extracellular acceptors are represented as cartoons. CbcL and ImcH are inner membrane-associated quinone oxidoreductases that transfer electrons to the PpcA-family cytochromes, which in turn are responsible for bridging the gap between the inner and outer membrane cytochromes ( 9 ). Prior to secretion, PgcA might also contribute to periplasmic electron transfer. The final steps of EET are warranted by porin-cytochrome complexes, cytochrome nanowires, and PgcA, which recruits metals and might synergistically work with these proteins to maximize the reduction of electron acceptors. The light gray cartoons represent metal oxides, attached to the disordered stretches of proline and threonine residues repeats. For simplification, the secretion domain of PgcA is not represented.",
"discussion": "Results and discussion PgcA and homologs establish a new class of cytochromes The AlphaFold model of PgcA shows that the triheme cytochrome possesses a fuzzy global arrangement with four structured domains linked by unstructured stretches ( Fig. S1 ). The first domain, classified as the secretion domain (residues 58–252, Fig. 1 A ), is preceded by a small amino acid sequence (Leu36-Ala37-Gly38-Cys39), designated lipobox motif, that is crucial for the correct translocation of PgcA out of the periplasm. In fact, the deletion of residues 37 to 39 inhibited this translocation ( 22 ). The motif is recognized by the prolipoprotein diacylglyceryl transferase (Lgt), which diacylates PgcA via Cys39’s sulfhydryl group. Immediately after, the signal peptide of PgcA is cleaved by the lipoprotein-specific signal peptidase (LspA/SpII) and the remaining N-terminal is acylated by the apolipoprotein N-acyltransferase (Lnt) ( 22 , 23 ). Finally, the lipoprotein is secreted by a Lol-like secretion pathway ( 18 , 22 ). During this process, PgcA is converted into its mature form, in which part of the secretion domain (residues 58–126, Fig. 1 B ) is cleaved ( 18 , 22 ). The exact residue at which PgcA is cleaved was previously determined by mass spectrometry, after its heterologous expression in Shewanella oneidensis , and correlated with earlier observations in Geobacter cell lysates ( 18 ). Nevertheless, the exact processing pattern of PgcA and the proteins involved in such mechanism can only be unequivocally identified after protein isolation directly from Geobacter cells. Curiously, according to the AlphaFold model of PgcA, the Ala126 residue (located in the frontier between the red and gray parts of the secretion domain - see Fig. 1 A ) at which the protein is supposedly cleaved is positioned at the end of a β-sheet, right before a loop, and thus highly accessible for the cleavage event. Figure 1 Structural features of PgcA. A , domain architecture of PgcA. The triheme cytochrome is composed by four domains, each indicated by different colors. This color code is respected in all figures. The secretion domain is divided in two parts ( red and gray ), which separate after protein secretion. The protein’s lipobox (LAGC), important for acylation signaling, is also highlighted. Cytochrome domains are represented in green (Domain 1), orange (Domain 2) and blue (Domain 3). The AlphaFold models of the different domains are represented as ribbon and surface. The heme axial ligands of each cytochrome domain are shown, with histidine and methionine axial residues in red and yellow , respectively. B , architecture of extracellular PgcA. After protein secretion and processing, PgcA is loose in the extracellular space, having different domains linked by unstructured stretches. C , amino acid sequence of PgcA’s domains. The CXXCH heme-binding motifs and distal axial ligands are underlined. The proline–threonine stretches that connect the different cytochrome domains are shown in black . Independently of the exact processing mechanism, the mature form of PgcA is diffusing freely in the extracellular space and contains three cytochrome domains ( Fig. 1 B ). These domains are linked by unstructured stretches, formed by repeats of proline and threonine residues ( Fig. 1 C ), which were previously shown to be important for binding with metal oxide surfaces ( 18 , 21 , 24 ). The proline residues restrict flexibility and impose structural conformations that confer higher affinity towards metal oxide surfaces while positioning the threonine hydroxyl groups for hydrogen bonding ( 18 , 21 , 24 ). Additionally, and based on the analysis of PgcA’s AlphaFold model, it is also conceivable to assume that these stretches provide enough flexibility for the different cytochrome domains to interact and exchange electrons, providing PgcA with functional advantages over other triheme cytochromes. In fact, in the majority of triheme cytochromes, the positions of the hemes are fixed within a certain structural frame ( Fig. S2 ) ( 25 ), thus limiting the distances at which the cytochromes can transfer electrons while providing other important functional features ( 26 ). Based on this data, we propose that PgcA is part of a new class of cytochromes, which we designated as microbial heme-tethered redox strings, whose structures resemble a beads-on-a-string arrangement, with the cytochrome domains working as beads and the unstructured stretches as strings. PgcA can adopt multiple conformations not only to place the cytochrome domains in favorable positions for efficient intramolecular electron transfer but also to stretch the distances at which it can reduce extracellular acceptors. In fact, if PgcA is fully stretched, the protein should be able to transfer electrons over a 20 nm distance. A similar arrangement has been observed in supramolecular heme tethering synthetic polymers ( 27 , 28 ), but it is unprecedented in biological systems. The amino acid sequence of PgcA was analyzed using Basic Local Alignment Search Tool (BLAST) to search for sequences with high homology. Interestingly, many of PgcA’s homologs possess the same predicted architecture ( Fig. S1 ), with multiple mono- and multiheme cytochrome domains linked by unstructured motifs, that can potentially stretch and transfer electrons over even larger distances. To gather insights into the functional mechanisms of PgcA, we used a biochemical deconstruction approach, a strategy also explored in the study of the dodecaheme cytochrome GSU1996 ( 29 ). This approach allows the study of each cytochrome domain of PgcA without dealing with the inherent difficulties of producing the full-length protein while cross-correlating and translating the results for the entire system. The cytochrome domains of PgcA are composed of α-helical structures with great stability Pure and homogeneous samples of each cytochrome domain were obtained after heterologous overexpression in Escherichia coli ( Fig. 2 A ). The cytochrome domains of PgcA contain one c -type heme-binding motif (CXXCH), around 65 residues, and a high degree of structural homology with the cytochrome OmcF from G. sulfurreducens ( Fig. S3 ) ( 30 ). The heme axial ligands of PgcA can be easily predicted by the AlphaFold model ( Fig. 1 ). While Domains 1 and 2 are distally coordinated by methionine residues (Met301 and Met392, respectively), Domain 3 is coordinated by a histidine residue (His491). Figure 2 Biochemical and spectroscopic features of the cytochrome domains of PgcA. A , size-exclusion chromatography elution profile after injection on a Superdex 75 Increase 10/300 GL molecular exclusion column, in 100 mM sodium phosphate buffer pH 8. The molecular weight of each domain is indicated. B , UV–visible spectral features in the reduced (colored) and oxidized ( black ) states. The local maxima of the UV-visible spectra are labeled. C , Far-UV CD spectral features. The local maxima and minima of each spectrum are highlighted, as well as the midpoint of unfolding transition (T M ) and enthalpy of unfolding (ΔH). The thermal unfolding profiles can be found in Fig. S4 . The arrows indicate the variation of the ellipticity with increasing temperature. D , redox titrations followed by visible spectroscopy. The squares and circles represent the data points in the reductive and oxidative titrations, respectively. The solid lines indicate the fittings of a Nernst equation to the experimental data, considering a one-electron reduction. The UV-visible spectra of the different domains ( Fig. 2 B ) display patterns similar to those shown by low-spin hexacoordinated hemes ( 31 ). The CD spectra ( Fig. 2 C ) show that the three cytochrome domains are mainly composed of α-helical secondary structural motifs, in correlation with the AlphaFold model predictions and with previous CD studies performed with the full-length protein ( 18 ). The percentage of secondary structural elements of each domain was estimated with the Beta Structure Selection (BeStSel) deconvolution method ( 32 ). The results have a reasonable correlation with the AlphaFold models ( Fig. S4 A and Table S1 ). The observed deviations can be attributed to the intrinsic limitations of the deconvolution process, which does not account for rare secondary structural elements and contributions from aromatic residues and large surface exposed cofactors, such as the heme groups in each domain ( Fig. S3 ) ( 33 ). The CD thermal melting profiles of each cytochrome domain ( Figs. 2 C and S4 B ) show that the proteins are thermoresistant, with melting temperatures of around 80 °C. This was independently confirmed by DSC ( Fig. S4 C ). PgcA contains an intramolecular redox chain Redox titrations followed by visible spectroscopy were performed for the three cytochrome domains ( Fig. 2 D ). No hysteresis was observed for any of the domains since the reductive and oxidative curves of each redox titration are superimposable. Thus, the redox process is fully reversible and no major structural rearrangements occur upon reduction or oxidation of the heme groups. The heme reduction potential values obtained for Domains 1, 2, and 3 were −48.4 ± 0.5 mV, −64.8 ± 0.7 mV, and −106.4 ± 0.4 mV, respectively. An extrapolation of these data to the full-length protein indicates that PgcA contains a redox-active window of 300 mV, ranging from −230 to +70 mV, and an apparent midpoint reduction potential of −71 mV. These values are thermodynamically compatible with the list of substrates that can be reduced by G. sulfurreducens and, in particular, by PgcA ( 9 , 18 ). Despite the high structural similarity between the different domains of PgcA ( Figs. 1 and S3 ), their reduction potential values are considerably distinct. There are several factors that modulate a heme reduction potential, namely the heme solvent exposure, the surrounding network of charged residues, and the nature or orientation of its axial ligands ( 34 ). In the case of PgcA’s domains, the observed differences can be explained by the nature of their heme axial ligands. The heme groups of Domains 1 and 2 have His-Met axial coordination and have higher reduction potential values compared to the one of Domain 3, which is axially coordinated by two histidine residues (His-His). On one hand, histidine residues are good electron donors and stabilize the heme’s oxidized state, resulting in lower reduction potential values ( 35 ). On the other hand, the sulfur atom of the methionine side chain is a good electron acceptor and stabilizes the iron’s reduced state, thus resulting in higher reduction potential values ( 36 ). The small difference in reduction potential between Domains 1 and 2 might be due to variations in the heme solvent exposure or by the surrounding network of charged residues. Compared to PgcA’s Domains 1 and 2, the homolog cytochrome OmcF - also possessing a His-Met axially coordinated heme - has a considerably higher heme reduction potential value (+180 mV). This is most likely a consequence of the existence of a fourth α-helix close to the heme, which further contributes to the shielding of OmcF’s heme from solvent exposure ( Fig. S3 ). Overall, the reduction potential values of the different PgcA’s cytochrome domains show that the protein can putatively establish an intramolecular thermodynamically favorable electron transfer chain by transferring electrons between its different hemes, as long as they are placed within 15 Å, according to Marcus’s theory of electron transfer ( 37 ). The AlphaFold model of PgcA ( Fig. S1 ) shows that these domains may be as far apart as 100 Å, but the flexible stretches that connect them may be essential to promote their contact and the concomitant interaction for electron transfer events. To test this hypothesis, we performed interaction studies followed by NMR, after the proper assignment of key heme substituent signals. NMR features of PgcA’s cytochrome domains NMR spectroscopy is an excellent technique to probe cytochrome’s properties, as it provides information on the heme’s spin state while delivering reliable fingerprints for the signals of the heme substituents and axial ligands in the reduced and oxidized states ( 9 ). Additionally, NMR is often used to study protein-protein or protein-ligand interactions extending from static to transient regime ( 38 ). In particular, most biophysical techniques are unsuitable to study the formation of transient complexes, such as those typically established by redox partners, which possess a short lifetime to ensure a fast turnover and a continuous electron flow ( 39 , 40 ). The most standard protocol used in NMR to map the interface between interacting molecules relies on chemical shift perturbation experiments, in which changes in the chemical environment of one molecule caused by interaction with other molecule(s) are monitored. In the particular case of cytochromes, since the interacting regions with other putative redox partners are necessarily located near the heme groups, the 1 H chemical shifts of the heme substituents are typically tracked. Thus, we started by acquiring 1D 1 H-NMR spectra of the cytochrome domains in the reduced and oxidized states, to assess the dispersion of the signals of the different heme substituents and axial ligands. The spectra show well-dispersed, narrow signals ( Fig. 3 ), indicating that the proteins are well-folded, in line with the results obtained by CD spectroscopy ( Fig. 2 C ). Figure 3 1D 1 H-NMR spectra of the cytochrome domains of PgcA. The spectra of the reduced ( upper ) and oxidized ( lower ) forms are represented. All spectra were acquired in 32 mM sodium phosphate buffer with NaCl (100 mM final ionic strength) at pH 7, 25 °C. The AlphaFold models of the different cytochrome domains are represented in ribbon with the putative heme axial ligands shown as sticks. Histidines and methionine residues are shown in red and yellow , respectively. The insets on the reduced spectra of Domains 1 and 2 highlight the assigned signals of the distal axial methionine residues. 2D 1 H-COSY spectra were used to distinguish between βCH 2 and γCH 2 protons. In general, low-spin hemes present narrower spectral regions compared to high-spin ones. In the reduced state, high-spin hemes present spectral regions ranging from −15 to 30 ppm, whereas low-spin hemes range from −5 to 10 ppm. In the paramagnetic oxidized state, high-spin cytochromes display considerably broad signals, with chemical shifts above 40 ppm, whereas low-spin cytochromes have narrower chemical shift windows, with the majority of the heme substituents signals located between −5 and 35 ppm. Therefore, from the observation of the 1D 1 H-NMR spectra of the three cytochrome domains in both redox states, it can be concluded that PgcA possesses three low-spin c -type hemes, as attested by the observed spectral widths in both redox states ( Fig. 3 ). This was independently confirmed by probing the temperature dependence of the heme methyl 1 H chemical shifts ( Fig. S5 ) ( 41 ). Based on this information, it can be further concluded that the cytochrome domains of PgcA are diamagnetic when reduced (Fe(II), S = 0) and paramagnetic when oxidized (Fe(III), S = 1/2). These features are rather convenient since the strategy to assign heme substituents is simpler and more straighforward for low-spin hemes in both redox states. In the diamagnetic reduced state, the 1 H NMR chemical shifts of the heme substituents are essentially affected by the heme ring-current effects ( 31 ), which are caused by the circular movement of electrons in the pyrrole rings ( 42 ). Consequently, the signals of each type of heme substituent ( Fig. S6 ) are located in well-defined regions of the spectrum ( 34 ). The heme propionate groups (13 1 CH 2 , 13 2 CH 2 , 17 1 CH 2 , and 17 2 CH 2 ) are the only exception, since they do not have particularly well-defined regions and are usually assigned resorting to connectivities with side-chain signals of residues located near the heme group. The first step of the assignment procedure in the reduced state is the analysis of the 2D 1 H-TOCSY spectrum, in which the connectivities between the 1 J-coupled thioether methines (3 1 H or 8 1 H) and thioether methyl groups (3 2 CH 3 and 8 2 CH 3 ) are identified ( Fig. S6 ). Following the identification of these connectivities, a 2D 1 H-NOESY spectrum can be used to establish spatial correlations between nuclei that are closer than 5 Å ( Fig. S6 ). In the paramagnetic oxidized form, in addition to the ring-current effects, the presence of an unpaired electron strongly contributes to the observed chemical shift of a heme substituent. In fact, the final observed chemical shift will strongly depend on the orientation and shape of the magnetic susceptibility tensor generated by the unpaired electron ( 43 ). Consequently, each type of heme substituent will not possess a well-defined region in the 1 H NMR spectrum and, hence, can be spread over the entire NMR spectral width. Alternatively, the 13 C chemical shifts of the heme methyl and propionate groups have typical regions that constitute solid starting points for their specific assignment. Therefore, in addition to the 2D 1 H-TOCSY and 2D 1 H-NOESY spectra, we also acquired a 2D 1 H, 13 C-HMQC spectrum in the oxidized state for each cytochrome domain ( Fig. S7 ). Apart from rare exceptions ( 44 ), monoheme c -type cytochromes possess His-Met coordinated hemes and it has been observed that the 7 1 CH 3 and 18 1 CH 3 heme methyls are typically more downfield shifted in the oxidized 1 H NMR spectrum than the 2 1 CH 3 and 12 1 CH 3 heme methyls. This pattern is also present in the oxidized spectrum of OmcF ( 30 ), as well as in those of Domains 1 and 2 ( Fig. 3 ). The observed pattern is a direct consequence of the fact that each of these pairs of heme methyls is attached to diametrically opposed pyrrole rings of the porphyrin ( Fig. S6 ), thus being differentially affected by the asymmetric distribution of the delocalized unpaired electron on the molecular orbitals of the porphyrin ( 41 ). Nevertheless, the chemical shift pattern observed in the oxidized spectrum of Domain 3 ( Fig. 3 ) is distinct because the heme axial coordination of this cytochrome domain is different (His-His) and, in these cases, the dispersion of the heme methyl signals is less conserved ( 43 ). Complementarily to the heme substituents, we also extended the assignment to some of the heme axial ligands of the cytochrome domains in both redox states. In the diamagnetic reduced state, the signals of the heme axial ligands are displaced towards the low-frequency region of the 1 H spectrum due to the heme ring-current effects. In proteins without heme cofactors, the side-chain proton signals of methionine residues (βCH 2 , γCH 2, and εCH 3 ) are found in the 1 to 3 ppm range, whereas those of the imidazole ring protons of histidines (ε 1 H, ε 2 H, δ 1 H and δ 2 H) are usually located in the 6 to 11 ppm range. In heme-containing proteins, when such residues axially coordinate the heme moiety, these signals are shifted to low-frequency by an average of 5 ppm. Consequently, on one hand, the signals of axially coordinated methionine residues display a characteristic pattern, consisting of a three-proton intensity signal at around −3 ppm from the εCH 3 group and up to four resolved one-proton intensity peaks of the βCH 2 and γCH 2 groups. On the other hand, axial histidines do not show any particular fingerprint in the high-field region of the 1 H NMR spectrum, because their signals are located in the protein’s signal envelope. The side-chain signals of the axially coordinated methionine residues of Domains 1 and 2 are highlighted in Figure 3 . In the oxidized state, there are typical spectral regions in the 2D 1 H, 13 C-HMQC spectrum for the αCH and βCH 2 signals of axial histidine residues ( Fig. S7 ) ( 45 ). The remaining side-chain protons of axial histidine and methionine residues, located even closer to heme iron, are usually broadened beyond detection as a consequence of the paramagnetic effect. Due to its three-proton intensity, the only exception is the methionine εCH 3 group, which despite its considerable broadening, can usually be detected in cytochromes with low molecular mass. Typically, this signal is observable between −9 and −25 ppm ( 46 ), and we were able to assign it for Domains 1 and 2 via 2D 1 H-EXSY spectra acquired with partially oxidized samples ( Fig. S8 ). These spectra show chemical exchange correlations between the signals of each nucleus at the reduced and oxidized states and were used to assign the εCH 3 group of the axial methionine residues, as well as to further confirm the assignment of the heme methyl groups in both redox states ( Figs. S8 and S9 ). The assigned signals are listed in Tables S2 and S3 for the reduced and oxidized states, respectively. PgcA cytochrome domains exchange electrons promiscuously To map the putative interacting regions between the different cytochrome domains of PgcA by chemical shift perturbation experiments, these must be conducted by keeping the molecules in the same oxidation state so that any change can be attributed to an interaction and not to a variation in the oxidation state. Based on this requirement, these studies were performed in the oxidized state, which is experimentally advantageous since sample manipulation in anaerobic conditions is not required and the chemical shifts of the relevant nuclei are more dispersed compared to the reduced state. Additionally, the 1D 1 H-NMR spectral features of the oxidized cytochrome domains ( Fig. 3 ) are distinct enough so that chemical shift variations throughout a titration can be easily monitored. The heme methyl groups are the best candidates to monitor chemical shift perturbations, not only because they are significantly shifted from the diamagnetic region, but also because of their three-proton intensity that facilitates their identification. NMR chemical shift perturbation experiments were carried out for the different combinations of cytochrome domain pairs by adding successive amounts of one cytochrome to the other and vice-versa. The analysis of the six independent titrations shows that all cytochromes interact transiently since their heme methyl signals undergo very slight chemical shift variation ( Fig. S10 ). The formation of low-affinity complexes between redox partners has been observed in other studies, and is crucial for fast turnover and continuous electron flow ( 47 , 48 ). To complement these experiments and to unequivocally confirm that the different cytochrome domains of PgcA exchange electrons upon interaction, we explored the distinct NMR spectral features of the domains in the reduced and oxidized states. By adding one cytochrome to the other in distinct redox states, and upon formation of the putative transient complex, we can observe the emerging signal features of the 1D 1 H-NMR spectrum of the cytochromes mixture and assess possible changes in their redox state ( 49 ). As for the NMR chemical shift perturbation experiments, six independent assays were performed for the possible combinations, by adding one oxidized cytochrome domain to a reduced one in anaerobic conditions ( Fig. S11 ). The results show that the cytochrome domains exchange electrons promiscuously until they reach an intermediate oxidation state, although at different extents. This can be quantified by comparing the area of the heme methyl signals of the two cytochrome domains in the intermediate redox state with those in the fully oxidized spectra. The calculated percentages show that, as expected, the cytochrome domains with more negative heme reduction potential values (−106, −65, and −48 mV for Domains 3, 2, and 1, respectively) transfer electrons to a greater extent than those with less negative heme reduction potential values ( Fig. 4 A ). In the opposite scenario, electron transfer is still observable but to a minor extent ( Fig. 4 A ). These variations in the extension of the reduction/oxidation of the different proteins correlate with the superposition of their redox-active windows ( Fig. 4 B ). This means that in full-length PgcA, electron transfer mostly follows the expected thermodynamically favorable route (Domain 3 – Domain 2 – Domain 1), but the cytochrome is capable of promiscuously transferring electrons between all domains, which can be advantageous during mineral reduction. Figure 4 Role of PgcA in the extracellular electron transfer pathways of G. sulfurreducens . A , summary of the electron transfer experiments between the different cytochrome domains of PgcA and different electron acceptors. The cartoons of Domains 1 ( green ), 2 ( orange ) and 3 ( blue ) are shown with their heme groups in red . The cartoons of akageneite, birnessite, Fe(III) citrate, and potassium chromate are shown in brown , purple , red , and yellow , respectively. The percentages correspond to the extent of the electron transfer reactions between each pair of domains. B , histogram comparison of the redox-active windows of G. sulfurreducens cytochromes and different electron acceptors. The redox-active windows of each cytochrome were determined from potentiometric redox curves considering the 1% to 99% range for protein reduction/oxidation. The cellular localization of the different cytochromes/redox-pairs is indicated in the bottom of the graph. The data used for the histogram can be found in Table S4 . The cytochrome domains of PgcA reduce several electron acceptors PgcA is a terminal reductase of extracellular Fe(III) and Mn(IV) oxides ( 18 ) and it might also be responsible for the reduction of other known extracellular electron acceptors of G. sulfurreducens . To assess this, we performed spectrophotometric analysis of the reduced cytochrome domains of PgcA in the presence of Fe(III) oxides, Mn(IV) oxides, Fe(III) citrate, and potassium chromate ( Fig. S12 ). Fe(III) oxides exist as a heterogeneous mixture of insoluble particles with different reduction potential values in Nature, which have the tendency to change as they are being reduced ( 50 , 51 ), meaning that these microorganisms require sophisticated mechanisms to cope with this challenge. Fe(III) oxides in the form of akageneite (β-FeOOH) present a redox-active window between −100 and +200 mV ( 50 ). Freshly prepared akageneite is usually fully oxidized and has a reduction potential above 0 mV ( 52 ). Upon addition of an excess of freshly prepared akageneite to Domains 1 and 2 in the reduced state, no differences in the visible spectra were observed ( Fig. S12 ). However, Domain 3 was able to reduce akageneite, as it can be seen by the differences in visible spectra of the cytochrome ( Fig. S12 ). In fact, the intensities of the typical bands of the reduced state – β (521 nm) and α (550 nm) bands – decrease upon addition of akageneite, as does the intensity of the Soret band, which also undergoes a blueshift from 417 to 408 nm. One of the most common forms of Mn(IV) oxides in soils and natural aquatic systems is birnessite (Na x Mn 2-x (IV)Mn(III) x O 4 , with x∼0.4), which presents a reduction potential of +612 mV ( 53 , 54 ). All cytochrome domains of PgcA were able to reduce birnessite ( Fig. S12 ). A similar spectral variation was observed for all the cytochrome domains upon the addition of Fe(III) citrate and potassium chromate ( Fig. S12 ), which are soluble forms of the Fe(III) and Cr(IV) cations and present reduction potentials of +370 mV ( 50 ) and +1350 mV ( 55 , 56 ), respectively. Overall, the results obtained indicate that the cytochrome domains of PgcA can reduce both soluble and insoluble forms of electron acceptors of G. sulfurreducens ( Fig. 4 ). Zacharoff and co-workers have previously shown that PgcA can bind Fe(III) oxides, but not Fe(II) oxides or mixed Fe(II)-Fe(III) minerals ( 18 ). By incubating PgcA with these metals and after recovery of the remaining soluble fractions, it was shown that this cytochrome selectively binds oxidized metals and releases them after their partial or complete reduction ( 18 ). These results, together with evidence from previous studies ( 21 ), indicate that the protein stretches of proline and threonine residues are responsible for such binding. Considering that our data shows that Domain 3 can reduce Fe(III) oxides, we decided to evaluate if the individual cytochromes domains of PgcA, in the absence of the repeats of proline and threonine residues, could also be involved in the binding of Fe(III) oxides. Using the methodology adopted by Zacharoff and co-workers ( 18 ), each cytochrome domain was incubated with Fe(III) oxides, and the resulting soluble fractions were recovered. The results show that only Domain 3 can bind Fe(III) oxides, to an extent of 20% ( Fig. S13 ), which correlates well with the fact that this domain was the only one capable of reducing this metal. Nevertheless, the amount of Fe(III) oxides bound to Domain 3 is considerably smaller than the one observed by Zacharoff and co-workers ( 18 ). In their study, the experiments were carried out with the three cytochrome domains linked by the flexible protein stretches of proline and threonine residues, and, in their case, the protein was completely bound to Fe(III) oxides ( 18 ). Therefore, our results partially correlate with their conclusions regarding the relevance of the disordered stretches of proline and threonine residues repeats in metal binding. Indeed, since only about 20% of the protein was bound to Fe(III) oxides in our experiments, it is conceivable to infer that the flexible protein stretches of proline and threonine residues are the main drivers of metal binding in PgcA."
} | 8,644 |
35036288 | PMC8752175 | pmc | 6,275 | {
"abstract": "Microalgae can produce biofuels, nutriceuticals, pigments and many other products, but commercialization has been limited by the cost of growing, harvesting and processing algal biomass. Nutrients, chiefly nitrogen and phosphorus, are a key cost for growing microalgae, but these nutrients are present in abundance in municipal wastewater where they pose environmental problems if not removed. This is not a traditional review article; rather, it is a fact-based set of suggestions that will have to be investigated by scientists and engineers. It is suggested that if microalgae were grown as biofilms rather than as planktonic cells, and if internal illumination rather than external illumination were employed, then the use of microalgae may provide useful improvements to the wastewater treatment process. The use of microalgae to remove nutrients from wastewater has been demonstrated, but has not yet been widely implemented due to cost, and because microalgae derived from wastewater treatment has not yet been demonstrated as a commercial source for value-added products. Future facilities are likely to be called Municipal Resource Recovery Facilities as wastewater will increasingly be viewed as a resource for water, biofuels, fertilizer, monitoring public health and value-added products. Advances in photonics will accelerate this transition.",
"introduction": "Introduction It is proposed that the increased use of microalgae to treat wastewater can benefit both the treatment of wastewater and the production of biofuels and various products from microalgae. More specifically, it is proposed that internal illumination should be used to support the growth of microalgae as biofilms. The reasons for these suggestions are briefly stated below, followed by more detailed discussions of the various topics mentioned. Why should microalgae be used in wastewater treatment? More rapid, complete and economical removal of pollutants. Nutrient recycling Residual nitrogen and phosphorus in wastewater effluent creates environmental problems, while the cost of growing microalgae can be decreased by utilizing nutrients in wastewater. Why should microalgae be grown as biofilms rather than as planktonic cells? Improved wastewater treatment By growing microalgae as biofilm, the retention of biomass is decoupled from the hydraulic retention time (HRT) allowing even microorganisms with cell generation times longer than the HRT to be retained and provide the amount of active biomass needed to treat wastewater as rapidly as possible. Biofilms are inherently more robust and can tolerate fluctuating environmental conditions, toxic chemicals and antibiotics. Decreased cost of harvesting and dewatering of biofilms as compared with planktonic cells. Why should internal illumination be used rather than external illumination of photobioreactors? Improved efficiency Photoinhibition can be avoided, and only light wavelengths used by photosynthetic microorganisms are supplied. Facilitates the growth of biofilms. Can be used in turbid solutions. More flexible reactor design. There is a high level of interest in developing biofuels as a replacement for fossil fuels, and microalgae have been viewed as a promising source of lipids for the production of biodiesel [ 1 – 4 ], or for the conversion of microalgae biomass to produce methane [ 5 , 6 ], or ethanol [ 2 , 4 ]. However, while an increasing list of value-added chemicals such as polyunsaturated fatty acids, carotenoids/pigments, antioxidants, biopolymers, and other nutraceutical and pharmaceutical compounds [ 2 , 7 , 8 ] are now commercially produced from microalgae, the economical production of biofuels from microalgae is still an unrealized goal [ 9 ]. Cost is the main constraint limiting large-scale production of renewable fuels from microalgae [ 10 ], but another important limitation is nutrients [ 11 , 12 ]. Not only is it costly to provide chemical nutrients to support microalgal growth, but the availability of phosphorus in particular could well become a limiting factor in the foreseeable future [ 6 , 7 ]. By using microalgae to treat wastewater, the cost of growing microalgae can be reduced as the nutrients in wastewater, chiefly nitrogen and phosphorus, can be recycled and used by microalgae [ 13 , 14 ]. The application of microalgae to treat wastewater has primarily focused on polishing wastewater to remove nitrogen and phosphorus after the majority of organic contaminants have been removed, but some studies have demonstrated that microalgae can be used instead of the activated sludge process as the main treatment for the removal of organic and inorganic contaminants [ 14 , 15 ]. The cost of microalgae production can be decreased if wastewater is used as a source of nutrients, and the cost of wastewater treatment can be reduced through the use of microalgae by improving the efficiency of the process and through the production of value-added by-products [ 9 , 10 ]. While the utilization of nutrients present in wastewater can reduce the cost of growing microalgae, the avoidance of environmental problems due to nutrients in wastewater effluent provides an even more important reason to consider the use of microalgae to improve wastewater treatment. Wastewater treatment challenges include land availability, cost and effectiveness [ 16 ]. Growing microalgae as biofilms, rather than as planktonic cells in solution, can facilitate the growth and harvesting of algal biomass and advances in photobiology can decrease the cost of growing microalgae, particularly as biofilms. The objective of this manuscript is to describe how photobiology can benefit wastewater treatment and lead to the development of a mature technology for the production of renewable energy and multiple biorefinery products from microalgae. The specific contribution to the literature made by this manuscript is to discuss how the diverse topics of wastewater treatment, microalgae, biofilms, internal illumination of photobioreactors, renewable energy, LEDs, photobiology, photoenzymes, materials science, artificial intelligence, robotics and engineering can all contribute to the development of future processes for the treatment of wastewater, recycling of nutrients and the production of bioproducts. Interdisciplinary research will be required to develop less expensive LEDs, inexpensive material suitable for the internal illumination of photobioreactors, the maximization of illuminated surface area for the growth of biofilm, the integration of microalgae into wastewater treatment, nutrient recycling and the production of value-added products. Researchers in these areas may be unaware of the collaborative opportunities in the use of microalgae in wastewater treatment and increased communication to stimulate research is the goal. The use of microalgae to treat wastewater, the growth of microalgae as biofilms and the use of internal illumination in microalgae photobioreactors are not new concepts, but what is new is increased awareness regarding recycling nutrients from wastewater, rapid innovation with a variety of renewable energy technologies, new discoveries in photobiology, decreasing costs for LEDs, an increased use of robotic devices to automate processes and increased interest in developing circular economies. This field is ripe for innovation and cross-discipline collaboration, and a goal of this manuscript to promote an awareness of the challenges and opportunities. Wastewater should be viewed as a resource rather than as an expense [ 17 , 18 ].",
"discussion": "Why are Biofilms Better than Planktonic Cells (a More Detailed Discussion)? Providing light to microalgae has been an enduring challenge both technically and economically [ 38 , 42 , 73 ]. Using microalgae to enhance the performance of wastewater treatment will face the same challenges of supplying light energy that currently limit the economical use of microalgae to produce biodiesel, ethanol, biogas or other products. Microalgae are most commonly grown as planktonic cells in open ponds (raceways) or in photobioreactors [ 4 ]. These planktonic cells are harvested by flocculation, filtration and centrifugation, and the resulting microalgae cell suspension is then dewatered to produce a more concentrated cell paste that is subsequently processed/extracted to make products. The cost of growing, harvesting and extracting microalgae currently prevents the use of microalgae to produce large volumes of low-cost products such as biofuels [ 31 ]. The cost of harvesting, concentrating and extracting lipids from microalgae biomass has been estimated to be 40–60% [ 1 , 74 ] of the cost of biodiesel, so that harvesting microalgae as biofilm can significantly decrease process cost. If light energy is supplied to microalgae growing as biofilms attached to optical surfaces immersed in wastewater, then the design of reactors used for wastewater treatment will not be constrained by the difficulty of supplying external illumination, and the need to periodically shut down the system to remove biofilm that is considered as unwanted when the goal is to grow planktonic cells [ 45 ]. As mentioned above, reactors currently used for the growth of microalgae for the tertiary treatment of wastewater or for the production of biofuels often employ shallow reactors/ponds that require large areas to accommodate the volume of wastewater to be treated or the mass of biofuel desired [ 39 ]. But if light is supplied via internally illuminated surfaces and not by external illumination, then the reactors can be of any depth/dimensions so a much smaller footprint/land area is required. Wastewater treatment facilities are typically located near population centers where the cost and availability of land can be a concern. Furthermore, if wastewater treatment plants in the future will produce an array of products derived from microalgae, then additional space may be required [ 2 , 4 ]. Accordingly, the use of shallow algae ponds for the treatment of wastewater will be difficult to implement at many facilities, and the most economical use of space of the wastewater treatment plant should be considered. The treatment of large volumes of water such as in wastewater treatment plants should be rapid because the treatment time determines how large the facility should be [ 19 ]. If microalgae biofilms improve the treatment efficiency, then smaller, less expensive treatment facilities can be constructed. By using internal illumination to supply light to microalgae the surface area of the biofilm is whatever it is designed to be and the limitation is the cost of optical surfaces, cost of electricity, harvesting and processing biomass. Biofilms are well documented to be more resistant to environmental contaminants, and antibiotics, than planktonic microbial cells [ 34 , 75 ]. Accordingly, maintaining a diverse microalgae/bacterial population, growing as biofilm, will improve the stability of the wastewater treatment process. Biofilms are retained in photobioreactors until harvested and so are best suited to enable rapid treatment of the maximum volume of wastewater since they allow survival of even microbial species with generation times longer than the hydraulic retention time of the reactor [ 66 ]. The composition of the biofilm communities in microalgae–bacteria biofilm wastewater treatment facilities will not be limited to only rapidly growing species; rather, a more complex microbial communities will form, each adapted to local conditions. Moreover, the mass of biofilm that can be retained within the facility can be controlled to maximize the efficiency and effectiveness of the removal of contaminants at the fastest possible flow rates [ 29 ]. Real-time monitoring of the concentrations of contaminants and the health of biofilms will be enabled by increasingly sophisticated and inexpensive analytical techniques such as single-cell multi-isotope nanoscale secondary ion mass spectrometry [ 76 ], photo-respirometry [ 71 ] and in situ metabolism [ 77 ] and the application of growth/process modeling [ 39 ] and artificial intelligence [ 78 ]. The goal of using internally illuminated surfaces to grow microalgae biofilm requires that the surface area of the light-emitting regions should be maximized; the subsequent harvest of biofilm from these surfaces is also an important consideration [ 36 , 63 , 74 ]. Porous light transmitting materials of natural or man-made origin could be favorable materials for the growth of microalgae biofilm [ 79 , 80 ] or nature-inspired structures such as leaves and trees [ 81 , 82 ] may be used. The harvesting of microalgae biomass from biofilm growing on internally illuminated surfaces could be accomplished using a robotic device that is equipped with cameras/optical sensors to locate biofilm and determine its readiness for harvest, and with multiple appendages that allow the robot to move about the wastewater treatment reactor, grasp biofilm-containing structures and mechanically collect the biofilm. When the biofilm storage capacity of the robot is reached, the biofilm/biomass can then be deposited in an appropriate location for further processing and the robot can return to harvest more biofilm. The harvesting of microalgae biomass is a major component in the cost of using microalgae, and by switching from harvesting planktonic cells to harvesting biofilm (that is already as concentrated as planktonic cells after dewatering), the harvesting process can be less complex and less costly. Moreover, by automating the task of biofilm harvesting by the use of robotic devices, processing costs can be further reduced [ 31 ]. Future research is required to obtain data that will allow a meaningful discussion of process cost. Scientists and engineers will identify how best to maximize the internally illuminated surface area within photobioreactors while simultaneously considering issues such as biomass harvesting method, wastewater treatment efficiency, maximum CO 2 fixation, materials of construction and cost. There are multiple configurations that a microalgae-focused municipal resource recovery facility might take and multiple cycles of design/build/test will be required to create the most functional and economic configurations [ 3 , 4 , 81 , 82 ]."
} | 3,577 |
26869180 | PMC4754335 | pmc | 6,276 | {
"abstract": "Human land use may detrimentally affect biodiversity, yet long-term stability of species communities is vital for maintaining ecosystem functioning. Community stability can be achieved by higher species diversity (portfolio effect), higher asynchrony across species (insurance hypothesis) and higher abundance of populations. However, the relative importance of these stabilizing pathways and whether they interact with land use in real-world ecosystems is unknown. We monitored inter-annual fluctuations of 2,671 plant, arthropod, bird and bat species in 300 sites from three regions. Arthropods show 2.0-fold and birds 3.7-fold higher community fluctuations in grasslands than in forests, suggesting a negative impact of forest conversion. Land-use intensity in forests has a negative net impact on stability of bats and in grasslands on birds. Our findings demonstrate that asynchrony across species—much more than species diversity alone—is the main driver of variation in stability across sites and requires more attention in sustainable management.",
"discussion": "Results and Discussion Variation in community stability Inter-annual variability of the total abundance of all species in a community (CV tot ) was much lower than the mean species-level variability (CV sp ) in both forests and grasslands ( Fig. 1 ). This stabilizing effect ranged from a 27% lower CV tot of forest bats to a 70% lower CV tot in resident forest birds and even a 72% lower CV tot in cover of grassland plants compared with the respective mean CV sp (arrows in Fig. 1 ). Community stability (CV tot −1 ) was significantly reduced in grasslands compared with forests, but the effect differed across taxa ( Supplementary Tables 1 and 2 ). For arthropods and birds, we found a 2.0- and 3.7-fold decrease, respectively, in community stability from forests to grasslands, whereas no differences were observed in bats. This destabilization was associated with lower asynchrony and higher level of CV sp in grassland arthropods and birds ( Supplementary Table 2 ). Plants had the most stable communities and showed the opposite trend, that is, grassland communities and populations were more stable and asynchronous than the forest understory vegetation. Note, however, that we excluded the tree and shrub layers, hence the most stable vegetation layers, from the analysis in forests. Land-use intensity gradients within forests and within grasslands showed relatively weak effects on community stability, which were only significant for grassland birds and forest bats ( Fig. 2 ). Land-use impacts on animal community stability were thus stronger for conversion of forests into open grassland than for gradual intensity variation, partly corresponding to a study on tropical birds 22 . Moreover, results for grassland plants—the taxon that has received most attention so far—may not fully represent the potential land-use responses of other organisms. Stability of arthropods, birds and bats negatively responded to conversion and/or intensity, whereas plants did not, supporting the idea that higher trophic levels can show increased sensitivity to land use and accelerate the losses of plant diversity 23 24 . Stability theories for consumers often focused on the roles of food-web structure, species mobility or body size 25 26 . While such food-web approaches often developed separately from merely plant-centred views 27 , insights from empirical studies across trophic levels—based on a common methodology and stability concept as in our study—may stimulate unified theories of stability in the future. Drivers of stability Generally, high instability (CV tot = σ / μ ) may arise from high standard deviation ( σ ) or from a low mean of the total abundance ( μ ), suggesting that either the community density or fluctuations are critical. In all communities in our study, σ was consistently more variable across sites than μ ( Supplementary Table 2 ), suggesting an important role of variance-driven instability. The negative land-use intensity effect on stability for forest bats ( Fig. 2 ) was driven by a significant decline in μ , which was not compensated by a decline in σ . The negative land-use effects on stability for grassland birds were, however, neither accompanied by significant changes in μ nor in σ ( Supplementary Figs 5 and 6 ). Gradual increases in land-use intensity indirectly affected stability through changes in asynchrony, diversity and abundance ( Fig. 3 ). Despite differences in the strength and significance of the three stabilizing pathways between taxa, land-use intensity generally decreased at least one of them ( Fig. 3 and Supplementary Figs 2 and 3 ). The strongest effects occurred via asynchrony and abundance for all taxa in forests, with an additional strong effect via diversity for forest bats. In grasslands, the strongest effects of land-use intensity on stability also occurred via asynchrony for all taxa, and additional effects via diversity were also consistent across all taxa. Land-use effects on animal community stability most likely also translate into changes in their ecosystem functions, for example, consumption-related processes such as parasitism, predation, herbivory, decomposition or pollination 28 . Hence, we also analysed herbivorous and carnivorous arthropods separately and found marked differences in how land-use intensity affects their stability. Forest land-use intensity reduced the stability of carnivores via asynchrony and abundance, whereas herbivore stability was affected via diversity ( Supplementary Fig. 4 ). Although grassland herbivore communities might become more unstable when fewer plant species are available, as shown in experimental grasslands 24 , the negative effects of land-use intensity on plant diversity did not translate into reduced stability of herbivorous arthropods in our study. Diversity decline in response to land use was not prevalent in all taxa, corresponding to earlier findings in the same sites 29 30 31 and elsewhere 32 . Hence, species diversity alone does not sufficiently capture critical changes in communities and ecosystems. In addition, different components of land use can have variable or even contrasting effects ( Supplementary Figs 2 and 3 ). In forests, harvesting of trees had the strongest negative impact on birds and bats, whereas non-natural tree species affected plants and arthropods most strongly. The proportion of non-natural trees also contributed to reduced bird abundance but increased plant and arthropod diversity. In contrast, the amount of dead wood with saw cuts was positively related to bird asynchrony. In grasslands, increases in bat abundance were driven by grazing intensity. Increased mowing and fertilization intensity as well as grazing intensity were associated with a lower diversity of plants, whereas mowing and fertilization increased the plants' asynchrony and abundance. Land-use intensity thus showed a negative impact on diversity, but this was compensated by asynchrony and abundance; thus, no net effect on stability was found. This finding corresponds to a study conducted in a grassland site in Michigan, where fertilization increased the asynchrony of plant communities and compensated for destabilizing diversity losses 11 . In contrast, in a Mongolian semiarid grassland plant community, the addition of nitrogen and phosphorous was found to be destabilizing for the plant community, whereas mowing had a stabilizing effect 17 . In a global meta-analysis, Hautier et al. 18 also showed that plant communities were destabilized when grasslands were fertilized, partly via a decreased asynchrony. Asynchrony significantly increased stability in all taxa and had by far the strongest impact on community stability in all taxa, except in forest bats where diversity had an equally strong influence. The primary importance of asynchrony was unaffected when an unweighted synchrony index was used (see Supplementary Tables 3 and 4 ). Diversity also contributed to stability in all grassland communities. Abundance had a stabilizing effect for birds, bats and plants in forests and for bats and arthropods in grasslands, but was destabilizing for forest arthropods ( Fig. 3 ). Asynchrony, diversity and abundance were often correlated ( Supplementary Figs 2 and 3 ). Diversity was positively correlated with asynchrony in forest bats and grassland arthropods, suggesting that common mechanisms may jointly affect both, or that compensatory dynamics increase with diversity. In contrast, plants, birds and bats in grasslands showed negative correlations between diversity and asynchrony. Different, complementary conservation and management actions in grasslands may thus be required when aiming to stabilize communities via both diversity and asynchrony. Interestingly, the decrease in asynchrony with higher diversity of grassland plants contrasts with the opposite trend reported along plant richness gradients in experimental 9 and naturally assembled grasslands 18 . Outlook Several studies found that the composition of species and their functional traits, functional performances or interactions can show marked changes with land use even when diversity remains unchanged 29 31 33 . Some of these functional traits can be relevant for stability, for example, thermal niches 33 or phenotypic plasticity 34 . Our results strongly support the view that other measures such as traits or inter-annual variability should complement diversity surveys to evaluate potential impacts of global change. We show that, despite the unequivocal positive diversity–stability relationship, diversity alone, without knowledge of the level of asynchrony, may be a poor indicator of community stability. Stabilization of communities and of their functional performance via diversity is an established goal for sustainable ecosystem management 2 . However, so far conservation assessments evaluating land-use impacts or the impact of land sharing versus land sparing between natural habitats and agriculture 35 are almost exclusively based on short-term diversity surveys. We suggest that future sustainable management concepts should also target and monitor processes and spatial habitat requirements that facilitate compensatory dynamics to promote inter-annual asynchrony as a key factor driving long-term stability."
} | 2,589 |
28068220 | PMC5222558 | pmc | 6,278 | {
"abstract": "Animals host multi-species microbial communities (microbiomes) whose properties may result from inter-species interactions; however, current understanding of host-microbiome interactions derives mostly from studies in which elucidation of microbe-microbe interactions is difficult. In exploring how Drosophila melanogaster acquires its microbiome, we found that a microbial community influences Drosophila olfactory and egg-laying behaviors differently than individual members. Drosophila prefers a Saccharomyces - Acetobacter co-culture to the same microorganisms grown individually and then mixed, a response mainly due to the conserved olfactory receptor, Or42b. Acetobacter metabolism of Saccharomyces- derived ethanol was necessary, and acetate and its metabolic derivatives were sufficient, for co-culture preference. Preference correlated with three emergent co-culture properties: ethanol catabolism, a distinct volatile profile, and yeast population decline. Egg-laying preference provided a context-dependent fitness benefit to larvae. We describe a molecular mechanism by which a microbial community affects animal behavior. Our results support a model whereby emergent metabolites signal a beneficial multispecies microbiome. DOI: \n http://dx.doi.org/10.7554/eLife.18855.001",
"introduction": "Introduction Multispecies microbial communities (microbiomes) influence animal biology in diverse ways ( McFall-Ngai et al., 2013 ): microbiomes modulate disease ( van Nood et al., 2013 ), metabolize nutrients ( Zhu et al., 2011 ), synthesize vitamins ( Degnan et al., 2014 ), and modify behavior ( Bravo et al., 2011 ). A central goal in host-microbiome studies is to understand the molecular mechanisms underpinning these diverse microbiome functions. Some aspects of microbial community function are the product of inter-species interactions ( Rath and Dorrestein, 2012 ; Manor et al., 2014 ; Gerber, 2014 ; Gonzalez et al., 2012 ). For example, microorganisms modulate the metabolomes of neighboring species ( Derewacz et al., 2015 ; Jarosz et al., 2014 ) and microbial metabolites (e.g., antibiotics) alter bacterial transcriptional responses ( Goh et al., 2002 ). Despite current understanding of microbial inter-species interactions in vitro, some of which has been elucidated in exquisite detail, the consequences of microbial interspecies interactions within host-associated microbiomes are just beginning to be explored experimentally. Insight into host-associated microbiome function has stemmed mostly from whole-microbiome [e.g., re-association of germ-free hosts with whole microbiomes ( Ridaura et al., 2013 ) and modeling microbiome function based on gene annotation ( Costello et al., 2009 )] or single-microorganism [e.g., re-association of germ-free hosts with a single microorganism ( Ivanov et al., 2009 )] studies. However, these approaches tend to reveal only limited insight into inter-species microbial interactions, which can provide hosts with essential services. For example, termite symbionts carry genes necessary for metabolism of different parts of complex carbohydrates ( Poulsen et al., 2014 ), yet their function has not been demonstrated in vivo; co-occurring human gut symbionts share polysaccharide breakdown products cooperatively ( Rakoff-Nahoum et al., 2014 , 2016 ), but the consequences of such interactions for the host are unknown; inter-species bacterial interactions protect Hydra from fungal infection ( Fraune et al., 2015 ), but the mechanism of host protection is unclear. The need to understand the effects of inter-species microbiome interactions motivated our current work. Attractive model systems in which to study the outcomes of inter-species microbial interactions for host biology would include a tractable host that harbors a simple multispecies microbiome. Here, we report the use of Drosophila melanogaster to study interactions in a simple microbiome and their consequences for host behavior. The Drosophila microbiome consists largely of yeasts, acetic acid bacteria, and lactic acid bacteria ( Chandler et al., 2011 , 2012 ; Broderick and Lemaitre, 2012 ; Camargo and Phaff, 1957 ; Staubach et al., 2013 ). Drosophila ingests microbiome members from the environment (e.g., fermenting fruit, [ Camargo and Phaff, 1957 ; Barata et al., 2012 ; Erkosar et al., 2013 ; Blum et al., 2013 ; Broderick et al., 2014 ]), a behavior posited as a mechanism for Drosophila to select, acquire, and maintain its microbiome ( Broderick and Lemaitre, 2012 ; Blum et al., 2013 ). Drosophila behavior toward environmental microorganisms has focused on yeasts ( Becher et al., 2012 ; Christiaens et al., 2014 ; Schiabor et al., 2014 ; Palanca et al., 2013 ; Venu et al., 2014 ). Yeasts attract Drosophila via ester production ( Christiaens et al., 2014 ; Schiabor et al., 2014 ), induce Drosophila egg-laying behavior ( Becher et al., 2012 ), and are vital for larval development ( Becher et al., 2012 ). Lactic and acetic acid bacteria produce metabolites (e.g., acids) that may repel Drosophila at high acid concentrations, while also inducing egg-laying preference for sites containing acetic acid ( Ai et al., 2010 ; Joseph et al., 2009 ). One motivation of our study was to analyze Drosophila behavior toward the yeast and bacteria that dominate the Drosophila microbiome. Yeast and bacteria are largely studied within separate Drosophila sub-disciplines, despite their shared habitat ( Broderick and Lemaitre, 2012 ). Yeasts serve as food, providing Drosophila vitamins, sterols, and amino acids ( Broderick and Lemaitre, 2012 ). Lactic and acetic acid bacteria are gut microbiome members ( Wong et al., 2011 ) promoting larval development ( Shin et al., 2011 ; Storelli et al., 2011 ), increasing resistance to pathogens ( Blum et al., 2013 ), inducing intestinal stem cell proliferation ( Buchon et al., 2009 ), and reducing adult sugar and lipid levels ( Newell and Douglas, 2014 ; Wong et al., 2014 ). Since microorganisms that are traditionally considered ‘food’ co-exist with those considered ‘microbiome’ in fruit fermentations and the two groups provide Drosophila with different resources, we hypothesized that Drosophila might detect a beneficial community via metabolites that are produced cooperatively by the desirable symbionts. Alternatively, Drosophila might detect a different metabolite as the signal for each symbiont. Fruit undergoes a well-characterized ripening process in which cell-wall degrading enzymes and amylases convert the firm, starchy tissue into soft, sugar-rich fruit ( El-Zoghbi, 1994 ; Abu-Goukh and Bashir, 2003 ; Mao and Kinsella, 1981 ). The high sugar content supports microbial colonization and fermentation by Drosophila -associated microorganisms, including yeasts, lactic acid bacteria, and acetic acid bacteria ( Barata et al., 2012 ; Barbe et al., 2001 ). Drosophila avoids ‘green’ fruit and is attracted to ‘overripe’ fruit ( Turner and Ray, 2009 ), yet it is unclear how Drosophila behavior is influenced by the dynamic multispecies fruit microbiome and its metabolic properties. To this end, we developed a model fruit fermentation system that afforded measurement of microbial populations, microbial metabolites, and Drosophila behavior. Here we demonstrate the importance of emergent microbiome metabolism—quantitatively different or unique metabolites produced by the microbiome, but not by any of its members in isolation—on behavior, suggesting that Drosophila larvae and adults benefit by behaviorally selecting a multispecies, interactive microbiome.",
"discussion": "Discussion Here, we have demonstrated how emergent properties of a microbial community—volatile profile, population dynamics, and pH—influence Drosophila attraction, survival, and egg-laying behaviors. Our study is the first to identify the consequences of microbe-microbe metabolic exchange on animal behavior and discovers additional microbial interactions that attract Drosophila for further mechanistic study ( Figure 1D ). Microbe-microbe metabolic exchange generates unique and quantitatively different volatiles from those resulting from individual microbial metabolism ( Table 1 and 2 , Figure 8 ). Acetobacter -generated acetate coupled to Saccharomyces -derived alcohols spawn diverse acetate esters ( Table 1 and 2 ). We hypothesize that more complex and diverse communities, comprising alcohol-producing yeasts, acetate-producing Acetobacter , and lactate-producing Lactobacillus , will generate a wider array of attractive esters ( Figure 8 ). The community of S. cerevisiae , A. malorum , and L. plantarum emitted higher levels of acetoin and attracted Drosophila more strongly than the co-culture of S. cerevisiae and A. malorum ( Figure 1D , Figure 5 ). Acetoin and 2,3-butanedione are formed by an α-acetolactate intermediate in bacteria and directly from acetaldehyde in yeast ( Chuang et al., 1968 ). We therefore hypothesize that communities of yeasts and bacteria may emit high levels of attractive acetaldehyde metabolic derivatives ( Figure 8 ). 10.7554/eLife.18855.053 Figure 8. Model of microbe-microbe metabolite exchange. Bolded are metabolites increased due to microbe-microbe interactions. DOI: \n http://dx.doi.org/10.7554/eLife.18855.053 Previous studies have found that yeasts alone can produce esters in high concentrations ( Becher et al., 2012 ; Christiaens et al., 2014 ; Schiabor et al., 2014 ). In this study, we found that S. cerevisiae produced low quantities of esters when grown alone. One explanation for the low ester production is that in contrast to previous studies that have used more complex media, we used an apple juice medium that is much lower in nitrogen content. Nitrogen content positively correlates with the yeast ester production ( Becher et al., 2012 ; Rollero et al., 2015 ). Our results suggest that environmental nitrogen availability might predict microbial ester production and Drosophila attraction. In high nitrogen environments, yeasts likely produce ester compounds and strongly attract Drosophila . However, in low nitrogen environments Acetobacter may be responsible for ester production and Drosophila attraction; Acetobacter may be capable of producing esters in low nitrogen conditions or may generate locally high nitrogen environments by assimilating nitrogen from yeast killed by its production of acetic acid. Future work should determine the relationship in wild fruit fermentations between nitrogen content and ester production by yeasts and bacteria. Drosophila behavioral studies have mostly focused on yeasts. Yeasts attract Drosophila and are the preferred substrate for Drosophila to lay eggs ( Becher et al., 2012 ). However, we find that Drosophila attraction toward the co-culture increases as yeast viability declines ( Figure 2 ). One reason why Drosophila might be attracted to the co-culture as yeast populations decline is that yeasts provide essential nutrients. As such, the lysis of viable yeast by Acetobacter may benefit Drosophila through the liberation of nutrients. An alternative explanation is that in their interaction with Drosophila, Acetobacter may have benefited by evolving to produce esters that in other contexts (e.g. high nitrogen environments) are produced by yeasts. The contribution of Drosophila -associated bacteria to Drosophila behavior is not as well understood as yeasts ( Venu et al., 2014 ). Our results suggest that non-yeast microorganisms, especially when grown in microbial communities, affect Drosophila behaviors. We reason that additional studies that couple chemical microbial ecology with Drosophila behavior will herald the discovery of additional microbe-influenced behaviors and microbial community-generated metabolites. This study demonstrates the coordination of ethanol synthesis and catabolism by S. cerevisiae and Acetobacter, respectively, and the role of ethanol in Drosophila behavior and survival. Non- Saccharomyces Drosophila microbiome members also make ethanol ( Ruyters et al., 2015 ) and diverse acetic acid bacteria catabolize ethanol, generalizing our findings to other microbial community combinations. Ethanol can have deleterious or beneficial fitness consequences for Drosophila depending on concentration ( Ranganathan et al., 1987 ; Azanchi et al., 2013 ) and ecological context ( Kacsoh et al., 2013 ). Our results are consistent with Drosophila using products of inter-species microbiome metabolism to detect a community that titrates ethanol concentration optimally for the host. Work that further dissects the consequences of acetic acid and ethanol concentrations on Drosophila biology and investigates other community-level metabolic profiles will be of interest to enrich the chemical and ecological portrait of the Drosophila microbiome. Drosophila egg-laying preference for the co-culture containing A. pomorum WT may provide a fitness tradeoff for the host. On the one hand, we observed that juice agar plates inoculated with the co-culture containing A. pomorum WT had fewer viable yeast cells and larvae developed more slowly, likely due to the lower vital nutrients (e.g. protein, vitamins) than would be available in the co-culture containing A. pomorum adhA . On the other hand, when exposed to environmental microbes, juice agar plates inoculated with the co-culture containing A. pomorum WT were not invaded by fungi, whereas the co-culture containing A. pomorum adhA was susceptible to fungal growth. This suggests that in more natural conditions the catabolism of ethanol into acetic acid, which delays larval development in the microbial community studied here (e.g. in a community with S. cerevisiae ), ultimately has a protective effect. Whether this is due to a direct elimination of pathogens or instead indirectly limits fungal competition, as has been shown for dietary yeasts and Aspergillus sp. ( Rohlfs and Kürschner, 2010 ) is unknown. Future work that more thoroughly dissects the Drosophila fitness tradeoffs that result from its association with different microbiomes is of interest. Our work raises questions about the consequences of the observed behavior on microbiome assembly and stability in the Drosophila intestine. Drosophila possesses specific and regionalized gut immune responses to the microbiome ( Lhocine et al., 2008 ; Ryu et al., 2008 ; Paredes et al., 2011 ; Costechareyre et al., 2016 ) implying a tolerant environment in which privileged microbiome members are maintained and reproduce in the Drosophila intestine. Other work suggests that Drosophila acquires its adult microbiome from exogenous sources, that adult microbiome abundance drops without continuous ingestion of exogenous microorganisms, and that the microbiome can be shaped by diet ( Chandler et al., 2011 ; Blum et al., 2013 ; Broderick et al., 2014 ). As such, a combination of internal mechanisms, exogenous factors, and host behavior likely sculpt the microbiome; determining the relative contribution of each will be important moving forward. Complicating our understanding of the contribution of these factors is the opaque distinction between ‘microbiome’ and ‘food’, since both are ingested from the environment ( Broderick, 2016 ). To dissect the formation and stability of the Drosophila microbiome, the fate of ingested microorganisms needs to be monitored and microbial intestinal replication needs to be surveyed as a function of Drosophila behavior, age, immune status, microbiome membership, and nutritional state [e.g. using synthetic diets without yeast; ( Shin et al., 2011 ; Piper et al., 2014 )]. In sum, our results support a model in which the Drosophila olfactory system is tuned to fruity (e.g., esters) and buttery (several acetaldehyde metabolic derivatives, such as 2,3-butanedione) smelling metabolites promoted by microbe-microbe interactions. We anticipate that accounting for microbial interactions in diverse host-microbe studies will lead to new insights into diverse aspects of microbial-animal symbioses."
} | 4,036 |
32141606 | PMC7496141 | pmc | 6,280 | {
"abstract": "Although cyanobacteria absorb blue light, they use it less efficiently for photosynthesis than other colors absorbed by their photosynthetic pigments. A plausible explanation for this enigmatic phenomenon is that blue light is not absorbed by phycobilisomes and, hence, causes an excitation shortage at photosystem II (PSII). This hypothesis is supported by recent physiological studies, but a comprehensive understanding of the underlying changes in gene expression is still lacking. In this study, we investigate how a switch from artificial white light to blue, orange or red light affects the transcriptome of the cyanobacterium Synechocystis sp. PCC 6803. In total, 145 genes were significantly regulated in response to blue light, whereas only a few genes responded to orange and red light. In particular, genes encoding the D1 and D2 proteins of PSII, the PSII chlorophyll‐binding protein CP47 and genes involved in PSII repair were upregulated in blue light, whereas none of the photosystem I (PSI) genes responded to blue light. These changes were accompanied by a decreasing PSI:PSII ratio. Furthermore, many genes involved in gene transcription and translation and several ATP synthase genes were transiently downregulated, concurrent with a temporarily decreased growth rate in blue light. After 6–7 days, when cell densities had strongly declined, the growth rate recovered and the expression of these growth‐related genes returned to initial levels. Hence, blue light induces major changes in the transcriptome of cyanobacteria, in an attempt to increase the photosynthetic activity of PSII and cope with the adverse growth conditions imposed by blue light.",
"conclusion": "Conclusions Our results show that the switch from artificial white to blue light had a much stronger effect on the gene expression profile, photophysiology and growth of Synechocystis sp. PCC 6803 than the switch to orange and to red light. In particular, PSII genes were upregulated in blue light, in agreement with the decreased PSI:PSII ratio of the cells. This photophysiological acclimation will improve the distribution of excitation energy between PSII and PSI, even though the low growth rate in blue light indicates that upregulation of PSII was insufficient to fully restore linear photosynthetic electron flow. Conversely, many ribosomal genes and other genes involved in protein synthesis were temporarily downregulated in blue light, concomitant with the transient decline of the growth rate. Hence, blue light not only results in a limited transfer of excitation energy to PSII and a low photosynthetic efficiency of PBS‐containing cyanobacteria, but also induces marked changes in their transcriptome to counter these adverse light conditions.",
"introduction": "Introduction Cyanobacteria play a key role in aquatic ecosystems and are widely hailed as the evolutionary ancestors of chloroplasts and thus are often used as model organisms to study oxygenic photosynthesis. Similar to eukaryotic photosynthetic organisms, cyanobacteria use the ubiquitous pigment chlorophyll a (Chl a ) in photosystem I (PSI) and photosystem II (PSII). Chl a absorbs in the blue part (peak wavelength at ~440 nm) and red part (680 nm) of the light spectrum (Engelmann 1882 , Kirk 2011 ). In addition, cyanobacteria deploy a diversity of phycobili‐pigments in large light‐harvesting antennae, known as phycobilisomes (PBS). These phycobili‐pigments include phycourobilin, phycoerythrobilin and phycocyanobilin, which have absorption maxima in cyan (495 nm), green (545 nm) and orange light (625 nm), respectively (Tandeau de Marsac 2003 , Six et al. 2007 ). Hence, cyanobacteria absorb blue light ≤450 nm. Yet, contrary to green algae and plants, PBS‐containing cyanobacteria have much lower rates of photosynthesis and growth in blue light than in the other light colors absorbed by their photosynthetic pigments (Lemasson et al. 1973 , Pulich and van Baalen 1974 , Wyman and Fay 1986 , Jørgensen et al. 1987 , Wilde et al. 1997 , Tyystjärvi et al. 2002 , Wang et al. 2007 , Singh et al. 2009 , Chen et al. 2010 , Choi et al. 2013 , Bland and Angenent 2016 , Luimstra et al. 2018 ). As a consequence, PBS‐containing cyanobacteria can be strong competitors in cyan, green or orange light absorbed by their PBS, but they are very poor competitors in blue light ≤450 nm in comparison to photosynthetic organisms with chlorophyll‐based light‐harvesting complexes (Luimstra et al. 2020 ). The cyanobacterium Prochlorococcus is an interesting exception. Prochlorococcus lacks PBS but instead uses divinyl‐chlorophyll‐based light‐harvesting complexes quite similar to green algae and terrestrial plants, and it performs very well in blue light as it is the most abundant cyanobacterium in the blue waters of the open ocean (Chisholm et al. 1988 , Flombaum et al. 2013 ). Why PBS‐containing cyanobacteria have a low photosynthetic efficiency in blue light is not yet fully resolved, but most likely it can be attributed to an imbalance of the light energy captured by PSI and PSII. In contrast to green algae and plants, PBS‐containing cyanobacteria invest much more of their Chl a in PSI than in PSII (e.g. Myers et al. 1980 , Fujita 1997 , Luimstra et al. 2018 ). As a consequence, PSI will absorb more blue light than PSII. Conversely, the PBS of cyanobacteria transfer most of their absorbed light energy to PSII (Joshua et al. 2005 , Mullineaux 2014 , Kirilovsky 2015 ). However, the phycobili‐pigments of PBS do not absorb blue light ≤450 nm (Grossman et al. 1993 , Tandeau de Marsac 2003 , Six et al. 2007 ). Hence, especially under light‐limited conditions, blue light provides sufficient excitation energy at PSI but insufficient excitation energy at PSII to sustain high rates of linear electron flow (Fujita 1997 , Solhaug et al. 2014 , Kirilovsky 2015 , Luimstra et al. 2018 , 2019 ). Several recent observations support the above‐mentioned explanation for the low photosynthetic efficiency of PBS‐containing cyanobacteria in blue light. At low light intensities, oxygen production rates of Synechocystis sp. PCC 6803 were much lower in blue than in orange and red light, whereas at high light intensities the maximum oxygen production rates were similar in all three light colors (Luimstra et al. 2018 ). Furthermore, several studies have shown that the PSI:PSII ratio of PBS‐containing cyanobacteria strongly decreases in blue light (Wilde et al. 1997 , El Bissati and Kirilovsky 2001 , Singh et al. 2009 , Luimstra et al. 2018 , 2019 ). Finally, a PBS‐deficient mutant of Synechocystis sp. PCC 6803 showed a similar low photosynthetic efficiency in both blue and orange‐red light, comparable to the PBS‐containing wild‐type in blue light (Luimstra et al. 2019 ). These observations are all consistent with the hypothesis that the low absorption of blue light by the light‐harvesting PBS, and hence the limited transfer of excitation energy to PSII, is the rate‐limiting factor for photosynthesis in blue light. In view of these strong photo‐physiological responses, we hypothesize that PBS‐containing cyanobacteria will display major changes in their gene expression profile when exposed to blue light. In particular, if blue light causes an imbalance between PSI and PSII, one may expect upregulation of PSII genes and downregulation of PSI genes. Furthermore, preferential excitation of PSI may lead to enhanced ATP production through cyclic electron flow without reduced nicotinamide adenine dinucleotide phosphate production (Allen 2003 , Munekage et al. 2004 , Yeremenko et al. 2005 ), which is likely to induce major shifts in the expression of many genes involved in the metabolic pathways that are dependent on the photosynthetic activity of the cells. Yet, although several studies have investigated how changing light conditions affect cyanobacterial gene expression (e.g., Hihara et al. 2001 , Gill et al. 2002 , Huang et al. 2002 , Billis et al. 2014 , Xiong et al. 2015 ), the transcriptome response of cyanobacteria to different colors of light has received much less attention (but see Singh et al. 2009 and Hübschmann et al. 2005 ). In this study, we therefore investigate how blue, orange and red light affect the gene expression profile of Synechocystis sp. PCC 6803. This PBS‐containing model cyanobacterium was grown in light‐limited chemostats and acclimated to ‘artificial white’ light (or more precisely, polychromatic light with an equal mix of blue, orange and red photons). When the chemostats reached steady‐state, the incident light was switched to monochromatic blue, orange or red light. The transcriptomes were analyzed just before and at several time points after this switch in light color, until a new steady‐state was reached. Hence, our transcriptome analysis allowed investigation of changes in gene expression associated with transient cellular processes as well as with long‐term acclimation to the three different light colors. In line with our hypothesis, the switch to blue light had a much stronger effect on the transcriptome than the switch to red and orange light, consistent with a cellular effort to restore the excitation balance between PSI and PSII. This striking difference in regulatory responses to different light colors adds further insight into the photosynthetic traits of cyanobacteria, which is of relevance for both biotechnological and ecological applications.",
"discussion": "Discussion Blue light alters photophysiology Our results show that Synechocystis sp. PCC 6803 produces much less biomass in blue light than in orange, red and artificial white light (Fig. 1 ). The low biomass production of PBS‐containing cyanobacteria in blue light is consistent with many previous findings (Wyman and Fay 1986 , Wilde et al. 1997 , Wang et al. 2007 , Singh et al. 2009 , Chen et al. 2010 , Choi et al. 2013 , Bland and Angenent 2016 , Luimstra et al. 2018 ). Blue light at wavelengths ≤450 nm is not absorbed by the PBS (Tandeau de Marsac 2003 , Six et al. 2007 ), which usually transfer most of their absorbed light energy to PSII. Blue light is absorbed by Chl a and carotenoids. Cyanobacteria tend to invest much more of their Chl a in PSI than in PSII (Myers et al. 1980 , Fujita 1997 ). Moreover, in cyanobacteria, only carotenoids of PSI appear to be involved in light harvesting, whereas carotenoids of PSII are involved in heat dissipation (Stamatakis et al. 2014 ). Hence, the low photosynthetic efficiency of cyanobacteria in blue light is commonly explained by the hypothesis that blue light causes an excitation imbalance between the two photosystems, with more light absorption by PSI than by PSII (Fujita 1997 , Solhaug et al. 2014 , Kirilovsky 2015 , Luimstra et al. 2019 ). This hypothesis also provides a rationale for the strong decrease of the PSI:PSII fluorescence emission ratio (Fig. 2 A; Fig. S1 A) and the decreased coupling of PBS to PSI, relative to PSII, after the switch from white to blue light (Fig. 2 B; Fig. S1 D). Similar physiological changes in response to blue light have been observed in previous studies (Tsinoremas et al. 1994 , Wilde et al. 1997 , El Bissati and Kirilovsky 2001 , Singh et al. 2009 , Luimstra et al. 2018 ), and reflect increased investments in PSII and/or decreased investments in PSI to compensate for the excitation imbalance between the two photosystems. The strong physiological response to blue light is also reflected in the transcriptome data, which showed that 145 genes responded to a switch from white to blue light whereas only a handful of genes responded to orange or red light (Fig. 3 ). In blue light several genes encoding structural PSII proteins were upregulated (Fig. 4 A), indicative of an effort to overcome the low amount of excitons reaching PSII relative to PSI. This response included an increased expression of the D1 and D2 proteins that form the core of the PSII reaction center, and the PSII core antenna protein CP47. Furthermore, we observed an increase in several genes related to PSII turnover, including 2 ftsH genes, hliB , hliC and lilA (Fig. 5 ). The ftsH genes encode proteins that are involved in the degradation of damaged D1 (Cheregi et al. 2007 ) and quality control of newly synthesized D1 (Komenda et al. 2006 ). The hliB and hliC genes encode high‐light inducible proteins that are suggested to stabilize PSII subunits during PSII repair (Promnares et al. 2006 , Yao et al. 2007 ). The lilA gene encodes a protein of the light‐harvesting protein family that associates with HliB and HliC (Kufryk et al. 2008 ). In contrast to this strong response of several genes related to PSII synthesis, none of the genes encoding PSI subunits were differentially expressed in blue light. Hence, the observed decrease in the PSI:PSII fluorescence emission ratio in blue light (Fig. 2 A; Fig. S1 A) seems to originate from an increased expression of PSII rather than a decreased expression of PSI. This is consistent with previous research using Synechocystis sp. PCC 6803, where blue light resulted in an upregulation of psbA encoding the PSII D1 protein, while expression levels of psaE encoding the Psa‐E subunit of PSI remained similar (El Bissati and Kirilovsky 2001 ). Furthermore, in blue light we observed an increase in the expression of nblA1 and nblA2 , encoding two PBS degradation proteins (Fig. 4 A). The gene slr1687 is presumed to encode another PBS degradation protein, NblB2 (Li and Sherman 2002 ), and was also upregulated in blue light (Fig. 4 A). Moreover, the expression of a molecular chaperone that is involved in stabilization of PBS (HtpG, see Sato et al. 2010 ) was downregulated (Fig. 5 ). Degradation of PBS might be induced because these antennae are not functional in blue light. Nitrogen‐rich PBS can constitute up to 50% of the total cellular protein content in cyanobacteria (Grossman et al. 1993 ), and their degradation provides building blocks for the production of other proteins. Indeed, the 77 K fluorescence spectra showed an increased decoupling of PBS from the photosystems in blue light (Fig. 2 C; Fig. S1 D). However, whole‐cell absorption spectra still showed considerable light absorption by phycocyanin at 625 nm relative to Chl a (Fig. 1 C), while previous research has shown that PBS degradation induced by NblA1 and NblA2 is accompanied by a decreased absorption by phycocyanin (Baier et al. 2001 ). Hence, although changes in gene expression appear to prepare the degradation of PBS and disassembly of the PBS may have been initiated, the degradation process is not yet fully activated in nutrient‐replete cells exposed to blue light. Blue light induces flocculation and pili formation Another interesting observation is the blue‐light induced upregulation of genes that play a role in phototactic motility (Fig. 5 ). In particular, our results show that the switch to blue light resulted in initial upregulation of pilA1 , known to be essential for the formation of Type‐IV pili and hence for twitching motility (Bhaya et al. 2000 ), and of pilA2 and sll1696 which are part of the same operon. Type IV‐based motility facilitates gliding of the cells over short distances on moist surfaces (Bhaya et al. 2001 ). Previous studies have indeed shown that blue light increases motility and negative phototaxis of Synechocystis sp. PCC 6803 (Ng et al. 2003 , Terauchi and Ohmori 2004 , Fiedler et al. 2005 ). After initial upregulation of the pilA1 ‐ pilA2 operon, the pilA9 , pilA10 and pilA11 ‐containing operon slr2015 ‐ slr2019 (encoding minor pili subunits) was also upregulated in blue light (Fig. 5 ). PilA9‐PilA11 have recently been shown to modulate the adhesive properties of Type‐IV pili and to enhance flocculation of the cells (Yoshihara and Ikeuchi 2004 , Panichkin et al. 2006 , Conradi et al. 2019 ). This was consistent with our observations, which showed increased flocculation and wall growth in the blue‐light chemostats (V.M. Luimstra, personal observation), a phenomenon that has also been described in previous studies (Enomoto et al. 2015 , Agostoni et al. 2016 ). Flocculation and negative phototaxis may be interpreted as a stress response, protecting Synechocystis cells from adverse light conditions imposed by blue light. Moreover, in Synechocystis sp. PCC 6803, type‐IV pili are essential for the uptake of extracellular DNA (Yoshihara et al. 2001 ) and flocculation of the cells might therefore also contribute to horizontal gene transfer (Conradi et al. 2019 ). Transient changes in gene expression related to reduced growth Remarkably, although genes associated with PSII and motility were upregulated in response to blue light for the entire duration of the experiment, many other genes were differentially expressed only temporarily after the switch to blue light and thereafter returned to their initial expression levels (Figs. 4 B, 5 ; Fig. S2 ). These transient changes in gene expression can be attributed to a temporary reduction of the growth rate after the switch to blue light, as illustrated by the declining cell numbers and biomass (Fig. 1 A,B). At steady state, the growth rate equals the dilution rate of the chemostat. Hence, after ~150 h, the cell numbers stabilized at a new steady state and the growth rate recovered to the same value as before the light switch. Low growth rates are usually associated with low ribosomal abundances (Scott et al. 2014 , Zavřel et al. 2019 ). The transient period of low growth rate in blue light was indeed accompanied by a temporary down‐regulation of 24 genes encoding ribosomal proteins and several other genes involved in transcription and translation (Fig. 5 ). Cellular metabolism is strongly ATP‐dependent, which explains why genes encoding ATP‐synthase were also temporarily downregulated during the transient period of reduced growth (Fig. 4 B). Furthermore, several genes encoding chaperones ( groES , groEL1 , groEL2 and htpG ), which commonly play an important role in the folding and stabilization of proteins, were also temporarily downregulated in blue light (Fig. 5 ). Interestingly, the only chaperone that was temporarily upregulated in blue light was the small heat‐shock protein HspA (Fig. 5 ), which plays a role in the stabilization of PSII and the thylakoid membrane (Nitta et al. 2005 , Sakthivel et al. 2009 ). Light sensing in cyanobacteria To allow acclimation to changes in their environment, microorganisms have developed highly efficient mechanisms to sense, transduce and respond to external signals. Cyanobacteria often use two‐component systems composed of a histidine kinase (Hik) and a cognate response regulator (Rre) (Appleby et al. 1996 , Mizuno et al. 1996 , Capra and Laub 2012 ). In total, Synechocystis contains 49 Hiks, of which six Hiks that have phytochrome‐like features are suggested to function as light sensors (hik1, hik3, hik24, hik32, hik35 and hik44; see Xu and Wang 2019 ). Of these six Hiks, only hik35 and hik32 were differentially expressed in our experiments. \n Hik35 ( slr0473 ) encodes the cyanobacterial phytochrome‐like protein Cph1 (Hughes et al. 1997 ) and appears to be involved in the regulation of at least 10 genes (Hübschmann et al. 2005 ). Hik35 and five Hik35‐regulated genes ( gifA , thrC , lilA , slr0373 , slr0376 ) were upregulated in our experiments after the switch to blue light, whereas one of the Hik35‐regulated genes ( tsf ) was downregulated. Tsf encodes the elongation factor TS which is involved in translation. The Hik35‐regulated genes slr0373 and slr0376 form an operon with slr0374 , and together these three genes represent the most strongly regulated genes in blue light (Fig. 3 ). The function of this operon is yet unknown, but it is known to respond to different stress conditions, such as iron deficiency, sulfur deficiency, nitrogen deficiency, high salinity, high light and oxidative stress (Singh and Sherman 2002 , Singh et al. 2004 ). The slr0374 gene encodes the highly conserved protein Ycf46. Inactivation of this gene reduces activity of the extracellular carbonic anhydrase EcaB, which indicates that it plays a role in the regulation of photosynthetic carbon fixation (Jiang et al. 2015 ). Hik32, also known as CcaS, is encoded by two hik32 genes ( sll1473 and sll1475 ; see Kondo et al. 2007 ), which were among the few genes that were significantly upregulated in orange and red light (Fig. 3 ). These two hik32 genes are interrupted by a transposase ( sll1474 ) in the Synechocystis strain (‘Kazusa’) sequenced in Cyanobase, but have been shown to form one gene in the original PCC 6803 strain that was used here (Okamoto et al. 1999 ). The two‐component system histidine kinase Hik32 and the cognate upstream response regulator CcaR ( slr1584 ) regulate the expression of cpcG2 ( sll1471 ) and the hypothetical gene sll1472 (Hirose et al. 2008 ), which were the two most strongly upregulated genes in orange and red light (Fig. 3 ). CpcG2 encodes a rod‐core linker protein that leads to the formation of atypical PBS that lack the allophycocyanin core and preferentially transfer energy to PSI, in contrast to the conventional PBS (containing CpcG1) that mainly transfer light energy to PSII (Kondo et al. 2005 , 2007 ). The phycocyanin‐containing PBS of Synechocystis sp. PCC 6803 absorb orange and red light very effectively (Tandeau de Marsac 2003 ), and usually transfer most of the absorbed light energy to PSII. Hence, the strong upregulation of cpcG2 in orange and red light might be interpreted as a functional adaptation to redistribute part of the light energy absorbed by PBS to PSI, presumably to maintain the excitation balance between PSI and PSII in orange and red light. It remains to be elucidated why cpcG2 and the hypothetical gene sll1472 were also upregulated after the transfer from white to blue light (Fig. 4 ; Fig. S2 ). Possibly, they might play a role in the observed uncoupling of PBS from the photosystems in blue light (Fig. 2 C; Fig. S1 D). None of the additional (blue light) photoreceptors (Fiedler et al. 2005 , Moon et al. 2010 ) showed a measurable transcriptional response to the imposed changes in light conditions. Comparison with other studies To the best of our knowledge, only one study has thus far focused on changes in the transcriptome of cyanobacteria in response to blue light (Singh et al. 2009 ). In their experiments, cells from a relatively dense Synechocystis culture acclimated to white light were inoculated in dilute batch cultures that were exposed to either blue or red light. Gene expression levels were measured only during the first 6 h of their experiments. Singh et al. ( 2009 ) observed a similar physiological response of Synechocystis sp. PCC 6803 as in our study, with a lower PSI:PSII ratio in blue light compared to red or white light. Some cautionary notes should be made before comparing their gene expression data with our results, however. In particular, Singh et al. ( 2009 ) quantified gene expression as the log 2 ratio of gene expression levels in red light relative to expression levels in blue light. Therefore, if gene expression was designated as, e.g., higher in red light it is uncertain whether this response actually reflected upregulation in red light, downregulation in blue light, or both. Furthermore, their batch cultures in red light showed a strong increase in cell numbers, while cell numbers in blue light barely increased during the first ~24 h. Hence, it is likely that differences in gene expression between red and blue light observed in their experiments reflect differences in light color, average light intensity perceived by the cells, and growth rate. This contrasts with our experiments, where we have used chemostats to separate transient effects caused by temporary changes in growth rate from persistent effects caused by differences in light color. Furthermore, we have used a dilute chemostat to separate effects of light color and light intensity. Despite these differences in experimental design, Singh et al. ( 2009 ) also report major changes in the expression of photosynthesis genes, with a higher expression of genes associated with PSII (and its synthesis) in blue light than in red light, including hliB , hliC , lilA and hspA , which are involved in PSII turnover and protection. Their Table S2 indicates that they also observed that the putative pilin genes pilA9‐pilA11 (related to motility) and the stress‐response operon containing slr0373 , slr0374 and slr0376 had higher expression levels in blue light than in red light. Furthermore, in accordance with the lower growth rate in blue light, they also observed lower transcript levels of genes encoding many ribosomal proteins, ATP synthase subunits and the chaperones GroES, GroEL1, GroEL2 after 3–6 h in blue light. In contrast to our findings, however, Singh et al. ( 2009 ) also observed a lower expression of several PSI genes in blue light than in red light. Furthermore, they report that expression of the motility genes pilA1 and pilA2 , and the PBS degradation genes nblaA1 and nblA2 was higher in red light than in blue light. This might be related to the lower light intensities used in their experiments, in line with results of Ogawa et al. ( 2018 ). Also, an increased expression of the cpcG1 gene encoding the conventional rod‐core linker protein of the PBS was observed in blue light while no data for the alternative cpcG2 gene were available from their experiments. In addition, contrary to our results, Singh et al. ( 2009 ) found large differences in cellular carbon and nitrogen metabolism between red and blue light. This might be related to differences in growth conditions between red and blue light in the experiments of Singh et al. ( 2009 ), where cells in blue light were still in initial lag phase and had not yet resumed growth. Hübschmann et al. ( 2005 ) investigated differences in gene expression between batch cultures of Synechocystis sp. PCC 6803 exposed to red light (652 nm) and far‐red light (734 nm). Red light of 652 nm is absorbed by Chl a and can also be absorbed by allophycocyanin in the core of the PBS, whereas far‐red light is absorbed only by Chl a and not by PBS (Lemasson et al. 1973 , Glazer and Bryant 1975 , MacColl 2004 ). Hence, we have theorized previously (Luimstra et al. 2018 ) that, similar to blue light, growth in far‐red light may also lead to an excitation imbalance between PSI and PSII. In line with this prediction, Murakami et al. ( 1997 ) showed that red light of 680 nm (which is not absorbed by PBS) resulted in a substantially lower PSI:PSII ratio than red light of 650 nm. Hübschmann et al. ( 2005 ) found that far‐red light of 734 nm indeed resulted in lower growth rates than red light of 652 nm. Moreover, many genes that were up‐regulated in blue light in our experiments were similarly up‐regulated in far‐red light in their experiments, including several genes related to PSII synthesis ( psbA2 , psbA3 , psbD2 , psbB ) and to PSII stabilization and turnover ( hliB , hliC , lilA , hspA ). Similarly, their Table S2 shows that the PBS degradation genes nblA2 and nlbB2 , and the genes slr0374 and slr0376 from the stress‐responsive operon described by Singh and Sherman ( 2002 ) were also upregulated in far‐red light. Furthermore, Hübschmann et al. ( 2005 ) found that the expression of more than 20 genes related to ribosomal proteins and several genes related to ATP synthase was lower in far‐red light than in red light, in line with the transient changes in gene expression when blue light temporarily reduced the growth rate in our experiments. This comparison indicates that many genes involved in photosynthesis and growth are similarly regulated in blue and in far‐red light, suggesting that these genes are indeed affected by the excitation imbalance between the two photosystems. However, some genes that were strongly upregulated or downregulated in blue light were not differentially expressed in far‐red light, indicating that these genes responded to blue light specifically. For example, hik35 encoding the cyanobacterial phytochrome Cph1 was upregulated in blue light in our study, but was not differentially expressed in red or far‐red light in the experiments of Hübschmann et al. ( 2005 ). And while blue light downregulated the expression of genes encoding the chaperones GroES, GroEL2 and HtpG in our experiments, these genes were upregulated in far‐red light in their experiments. Hübschmann et al. ( 2005 ) also found that most pilin genes were not regulated or even downregulated in far‐red light. By contrast, in our experiments blue light induced upregulation of several pilin‐genes involved in motility and flocculation of cells (Fig. 5 ), in line with other research showing that blue light has distinct effects on cellular motility and flocculation (Ng et al. 2003 , Terauchi and Ohmori 2004 , Fiedler et al. 2005 , Savakis et al. 2012 , Enomoto et al. 2015 , Agostoni et al. 2016 )."
} | 7,307 |
25054332 | PMC4108314 | pmc | 6,283 | {
"abstract": "Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae , whose expression is controlled by sigma factor σ E . In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5′ untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the σ E -regulon and biofilm formation in V. cholerae . In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.",
"introduction": "Introduction \n Vibrio cholerae inhabits aquatic environments and when it enters the human intestine, e. g., through ingestion of contaminated food or water, it causes the severe diarrheal disease, cholera. Vibrios are shown to form biofilms on zooplankton, insects and intestines [1] – [5] . Compared to planktonic cells, bacteria within biofilms are more resistant to stress conditions, e. g., osmotic and oxidative stress, acidity, antibiotics exposure and immune clearance [6] – [12] . Biofilm structures are constructed of and maintained by biofilm matrix components [13] . In V. cholerae , formation of biofilm requires production of exopolysaccharide (VPS) and the biofilm matrix proteins RbmA, RbmC and Bap1 [14] – [18] . These matrix proteins appear to be involved at particular steps during the biofilm formation process. RbmA is involved in the initial cell-cell adhesion step and serves as a tether, forming flexible linkages between cells and the extracellular matrix [18] , [19] ; Bap1 facilitates adherence of the developing biofilm to surfaces; and the heterogeneous mixtures of VPS, RbmC and Bap1 appear to form envelopes to encase the cell clusters [18] . Without RbmC, incorporation of VPS through the biofilms is significantly reduced, suggesting an essential role for RbmC in maintaining the mature biofilm structure [18] . To date, studies on the regulation of biofilm formation have been mainly focused on VPS synthesis. A complex regulatory network controls transcription of the vps gene in response to multiple environmental signals, such as signals from quorum-sensing bacterial autoinducers [20] , polyamines [21] , [22] , nucleosides [23] , [24] , indole [25] and nutrient scarcity [26] . Recently, glucose-specific enzyme IIA has also been shown to regulate biofilm formation through binding to a carbon storage regulator homolog MshH, demonstrating a link between the phosphoenolpyruvate phosphotransferase system and biofilm formation [27] , [28] . In contrast to the vast body of knowledge about VPS regulation, very little is known about regulation of the matrix proteins (RbmA, RbmC and Bap1). Fong et al [29] has demonstrated the involvement of two factors: the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and a transcriptional regulator VpsR. While VpsR positively regulates transcription of the rbm genes, cAMP-CRP appears to negatively regulate rbm expression, both mediated by and independently of VpsR [29] . In the past decade, an increasing body of evidence has highlighted the important and complex roles of small regulatory RNAs (sRNAs) in bacterial physiology and pathogenesis [30] , [31] . Many sRNAs are produced in response to specific environmental signals/stresses. They act by base-pairing with target sequences, resulting in up- or down-regulating gene expression through modulating the translation or the turnover of target mRNAs (see review [32] ). This mechanism of regulation often requires the RNA chaperone protein Hfq that facilitates base pairing between sRNAs and their target mRNAs [33] , [34] . In Vibrio , a σ E -dependent sRNA, VrrA, has been shown to be induced by envelope stress and to repress the outer membrane proteins OmpA and OmpT through base pairing to the 5′ untranslated regions (UTR) of the corresponding mRNAs. When the OmpA level decreases, envelope stress is reduced by releasing outer membrane vesicles (OMVs) [35] , [36] . These OMVs further protect bacteria against environmental hazards such as UV damage [37] . Using the infant mouse model, VrrA was demonstrated to attenuate V. cholerae virulence [35] , which could be partially explained by the VrrA-mediated down-regulation of TcpA, a major V. cholerae virulence factor essential for host colonization. In this study, we provide evidence that VrrA down-regulates the biofilm matrix protein RbmC by base-pairing with the 5′-UTR of rbmC mRNA. Because RbmC is essential for maintaining the mature structure of biofilms, this VrrA-mediated suppression of RbmC might be an additional mechanism of biofilm regulation in V. cholerae .",
"discussion": "Discussion \n V. cholerae transits between fundamentally different habitats the aquatic environment and the human digestive tract. Such transitions require rapid acquisition and integration of environmental cues in order to coordinate adequate genetic programs and adapt to the new niche. One such adaptation program involves the switch between a planktonic, motile lifestyle and a biofilm-based sessile lifestyle. To date, numerous regulator proteins have been found to affect biofilm formation in V. cholerae , such as those described in the Introduction . Results from this study add a new class of regulators, sRNAs, as a direct regulator of a biofilm matrix component. Through down-regulation of RbmC, VrrA weakens the stability of the mature biofilm structure and might therefore facilitate dispersal of bacteria from a sessile to a planktonic life style. In addition, because expression of VrrA is controlled by sigma factor σ E , VrrA serves as a molecular link between the σ E -regulon and biofilm formation in V. cholerae . Several sRNAs have been shown to be involved in biofilm formation in E. coli and Salmonella , e. g. OmrA/B [41] , McaS [42] , [43] , RprA [44] and GcvB [42] . In contrast to VrrA, these sRNAs do not target biofilm matrix components directly, instead they target biofilm master regulators such as CsgD, which in turn regulates biofilm components. This generates a hierarchical regulatory network and enables csgD mRNA to serve as a hub for complex signal integration via multiple sRNAs [45] , [46] . Similarly in Vibrio , sRNAs Qrr1-4 and CsrB/C/D regulate the biofilm master regulator HapR or the regulatory molecule cyclic di-GMP (through diguanylate cyclase) [47] , [48] , and thus are indirectly involved in biofilm formation. VrrA belongs to a growing family of sRNAs that regulate multiple targets [48] , [49] . VrrA uses unique pairing regions to differentially regulate different mRNA targets. Compensatory base pair change experiments revealed that residues C 100 U 101 U 102 (numbers relative to the +1 transcriptional start site) in VrrA are essential for base-pairing with rbmC mRNA, while those required for the regulation of ompT mRNA are G 73 C 74 U 75 in VrrA [36] . In addition to the target-specific regulating regions in VrrA, dependency on the chaperon protein Hfq differs among mRNA targets as well. Although deletion of hfq abolishes the interaction between VrrA and ompT mRNA, Hfq is not absolutely required for the regulation on ompA \n [35] or rbmC mRNAs (this study). The observation that OmpA and RbmC levels were elevated in the Δ hfq strain and that VrrA could only partially repress this elevated expression suggests that additional sRNAs are involved in the regulation. The combination of target-specific regions in VrrA and differentiated requirement of Hfq allows VrrA to modulate multiple targets differentially. According to the RNAhybrid prediction, as shown in Fig. 2 , A 91 C 92 U 93 C 94 C 95 U 96 in VrrA base pairs to the potential SD sequence ( AGGGAGU ) of rbmC . We therefore expected to see the most drastic change in RbmC level in strains expressing VrrA M7 (substitution of A 91 C 92 U 93 C 94 C 95 U 96 with U 91 G 92 A 93 G 94 G 95 A 96 ) and VrrA M8 (substitution of A 91 C 92 U 93 with U 91 G 92 A 93 ). However, our results showed that VrrA M9 and VrrA M10 , which base pairs to the region upstream of the SD sequence, had more impact on the regulation of RbmC. This unexpected result might be due to the fact that the SD sequence was predicted based on the consensus sequence and therefore might not be the exact SD site. Future studies using e. g. toeprint analyses will hopefully identify the actual interaction site(s) between VrrA and rbmC mRNA. Nevertheless, the present results from the compensatory base pair substitution experiment demonstrate that there is a direct interaction between VrrA and rbmC at the region upstream of the putative SD sequence ( Fig. 4B ). It is noteworthy that there are only a few functional homologs to VrrA in other Gram-negative bacteria. One such example is the MicA sRNA in Salmonella and E. coli \n [50] , [51] . Both MicA and VrrA are σ E -dependent and are capable of down-regulating multiple outer membrane proteins by base-pairing mechanisms [35] , [52] . Interestingly, Kint et al [45] observed that MicA in Salmonella was involved in biofilm formation, although the molecular mechanism remains unknown. Systematic searches for MicA targets using bioinformatics prediction tools have not identified yet any biofilm-related genes. Future work will be needed to examine possible interactions between MicA and Salmonella biofilm components such as curli and fimbriae. In summary, VrrA is the first example of an sRNA molecule that directly targets expression of a biofilm matrix component. Given the similarities between VrrA and its homologs in other Gram-negative bacteria, it is plausible that similar direct regulation exists in other bacteria as well. Because VrrA weakens the stability of the mature biofilm structure, strategies directed towards mechanisms or levels of sRNAs to disturb bacterial biofilm formation may potentially be used to combat biofilm-related infections. Furthermore, in our earlier studies, we showed that the TcpA, one of the colonization factors of V. cholerae , was down-regulated by VrrA (Song et al. 2008). In this study, we demonstrated that the expression of one of the extracellular matrix proteins, RbmC that is important for the biofilm formation by V. cholerae was modulated by VrrA. We hypothesize that at the later stage of V. cholerae infection in the host, bacteria can move away from the epithelial surface and into the fluid-filled lumen of the intestine. During this time, the bacteria may undergo a switch from attachment to the epithelial surface to detachment. This process may be associated with up-regulation of VrrA. We suggest that this transition prepares the bacteria to leave the intestine, for survival in the environment, and for eventual transmission to a new host. This process might be orchestrated by VrrA that can modulate expression of both a colonization factor (Tcp) and attachment factor (RbmC)."
} | 2,806 |
23425591 | null | s2 | 6,284 | {
"abstract": "Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)-mediated bacterial migration leads to the formation of a fibre-like Psl matrix. Deletion of T4P in wild type and flagella-deficient strains results in loss of the Psl-fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air-liquid interface. Bacteria lacking T4P-driven twitching motility including those that still express surface T4P are unable to form the Psl-fibres. Formation of a Psl-fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl-fibres are likely formed by Psl released from bacteria during T4P-mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P-driven twitching motility. Furthermore, a radial-pattern Psl-fibre matrix is present in the middle of biofilms, a nutrient-deprived region. These imply a plausible model for how bacteria respond to nutrient-limited local environment to build a polysaccharide-fibre matrix by T4P-dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings."
} | 353 |
23930134 | null | s2 | 6,287 | {
"abstract": "The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field."
} | 286 |
25784647 | PMC4363858 | pmc | 6,288 | {
"abstract": "Increased plant productivity and decreased microbial respiratory C loss can potentially mitigate increasing atmospheric CO 2 , but we currently lack effective means to achieve these goals. Soil microbes may play critical roles in mediating plant productivity and soil C/N dynamics under future climate scenarios of elevated CO 2 (eCO 2 ) through optimizing functioning of the root-soil interface. By using a labeling technique with 13 C and 15 N, we examined the effects of plant growth-promoting Pseudomonas fluorescens on C and N cycling in the rhizosphere of a common grass species under eCO 2 . These microbial inoculants were shown to increase plant productivity. Although strong competition for N between the plant and soil microbes was observed, the plant can increase its capacity to store more biomass C per unit of N under P. fluorescens addition. Unlike eCO 2 effects, P. fluorescens inoculants did not change mass-specific microbial respiration and accelerate soil decomposition related to N cycling, suggesting these microbial inoculants mitigated positive feedbacks of soil microbial decomposition to eCO 2 . The potential to mitigate climate change by optimizing soil microbial functioning by plant growth-promoting Pseudomonas fluorescens is a prospect for ecosystem management.",
"discussion": "Discussion The capability of plants and soil microbes to successfully sequester atmospheric C in terrestrial ecosystems largely depends on plant productivity along with microbial decomposition and mineralization feedbacks within the rhizosphere 8 10 11 12 . Advancing current efforts to mitigate effects of climate change could minimize harmful effects of elevated CO 2 1 26 . Here, for the first time, we show that addition of a microbial inoculant has the potential to promote plant productivity while mitigating positive feedbacks of microbial decomposition to increased plant C inputs that typically accompany eCO 2 . Our results also demonstrated that P. fluorescens inoculation led to increased plant tissue C:N under eCO 2 , resulting in an increased capacity to store C per unit of N in plant tissue. Therefore, this soil microbial inoculant may be a useful tool to mitigate climate change. The results from this study are consistent with numerous field and growth chamber experiments showing that P. fluorescens inoculants can increase plant production under ambient CO 2 14 15 31 , and show that these largely stimulating effects could be additive when combined with eCO 2 . Although it was impossible to exclude potential fertilization effects on plant available N through bacterial cell addition, the increases in total soil N pool by bacterial cell addition (0.016%) is likely negligible in comparison with the increase in plant growth by an average of 42%. In addition, chemical adjustments to litter C:N may contribute to reduced quality and decomposability of plant litter under eCO 2 9 34 which may reduce soil decomposition rates. In comparison to our previous study, overall plant C:N ratios during the rapid vegetative growth stage were lower than during the reproductive stage 35 . Plant roots play an essential role in regulating acquisition of soil nutrients. Nevertheless, little is known about the relationship of root functional traits with plant N use strategies. Root surface area is generally correlated to plant nutrient uptake rates 36 . P. fluorescens inoculants significantly increased root surface area under eCO 2 ( Table S1 ), suggesting that P. fluorescens inoculants could enhance the potential for plant roots to acquire N under N-limited eCO 2 condition. Moreover, our results demonstrated that root surface area positively correlated with plant C:N ratio across experimental treatments ( Figure 2a ), further indicating that the plants' ability to acquire N from the soil could be influenced by soil N availability. Coupled C and N processes in the rhizosphere play a critical role in maintaining the sustainability of ecosystems 34 37 . Recent studies suggest that plant productivity slows when plant N demand decouples from soil N cycling under climate change or other ecological disturbances 38 39 . In our previous study conducted in the same ecosystem as this present work, we found that eCO 2 increased microbial biomass N immobilization and decreased soil N availability 19 . Likewise, this study revealed that eCO 2 significantly decreased soil enzyme C:N stoichiometry ( Table S1 ), indicating greater microbial demand for soil N under eCO 2 40 . N limitation could ultimately dampen ecosystem C sequestration in terms of the eCO 2 fertilization effect on plant productivity 10 34 . However, P. fluorescens inoculant did not directly affect soil enzyme C:N stoichiometry ( Table S1 ). Moreover, the negative relationship between plant biomass C:N and enzyme C:N ( Figure 2b ) suggests that plants can continue to grow through increase in their capacity to store C per unit of N in response to changes in soil N availability, which is mediated by soil microbial activities. It is well known that plants can alter their N uptake rates to cope with plant physiological and environmental changes 41 42 . By using the 15 N isotopic method, we observed that eCO 2 increased the importance of N mineralized from SOM, indicating that the positive effects of eCO 2 on soil N enzyme activities increased soil N availability ( Figure 3a and Table S1 ). However, P. fluorescens inoculants had no detectable effects on δ 15 N values of plant biomass ( Figure 3a ) or on soil N enzyme activities ( Table S1 ). This suggests that P. fluorescens does not facilitate N mineralization under eCO 2 conditions. We note that N in P. fluorescens cells is mostly in organic form with the δ 15 N value of 4.2‰ (the δ 15 N value of organic N in SOM is 587.5‰). If plants took up mineralized N from dead P. fluorescens cells, the δ 15 N value of plant biomass should be lower than plant uptake. However, even if all bacterial N was absorbed by plants, the N in P. fluorescens cells would only contribute from 0.8% to 2.2% of the total plant N pool. In addition, the δ 15 N value of organic N corrected by bacterial cells (587.4‰) was still much higher than inorganic N (445.7‰). eCO 2 -induced rhizosphere priming effects and subsequent microbial N mineralization could influence the magnitude of plant growth 8 21 . Previous studies have identified several plant and microbial traits related to RPE 8 21 43 , but the direct evidence of priming-related effects on plant N availability has not been well documented. Our results, for the first time, clearly demonstrated that priming of SOM decomposition was positively related to plant N availability ( Figure 3b ), suggesting that priming made soil N more available to the plant for uptake. Increased root length was also observed in this study as an important root functional trait related to plant N uptake adaptations associated with microbial plant growth-promoting properties under eCO 2 conditions. These results add to a growing body of evidence that plants could increase N availability through rhizosphere priming and development of root systems to alleviate nitrogen limitation under eCO 2 8 9 21 43 . P. fluorescens inoculants and eCO 2 were expected to increase plant C inputs to soil ( Figure 4a ). However, a synergistic effect of bacteria and eCO 2 on plant-derived C was not observed ( Figure 4a ). This may be due to the use of planting pots which may have constrained root growth in this experiment. Results from a meta-analysis suggest that CO 2 -induced increases in belowground biomass are stronger in plants grown in open fields relative to closed pots 9 . In spite of higher rates of new C inputs, P. fluorescens inoculants and eCO 2 demonstrated contrasting effects on heterotrophic respiration due to microbial activities. eCO 2 increased mass-specific microbial respiration ( Figure 4b ) 35 , consistent with previous observations of climate-induced positive feedbacks 12 40 . P. fluorescens inoculants did not change mass-specific microbial respiration under ambient CO 2 but mitigated positive microbial feedbacks under eCO 2 conditions ( Figure 4b ). Thus, these findings suggest that P. fluorescens inoculants may potentially decrease soil C losses via heterotrophic respiration. Our results indicate that P. fluorescens inoculants may optimize soil microbial functioning and potentially be implemented as a strategy for increasing plant productivity while mitigating positive feedbacks of microbial decomposition to eCO 2 . If the benefits of P. fluorescens inoculants can be scaled from the growth chamber and applied in natural ecosystems in a high-CO 2 world, the potential for terrestrial C sequestration may increase to mitigate rising atmospheric CO 2 . Further assessment is needed to extend these findings to field experiments and to formulate economical methods of inoculation for field deployment. Additional experiments should be performed to assess rhizosphere colonization by P. fluorescens inoculants across a range of plant species."
} | 2,277 |
25784647 | PMC4363858 | pmc | 6,288 | {
"abstract": "Increased plant productivity and decreased microbial respiratory C loss can potentially mitigate increasing atmospheric CO 2 , but we currently lack effective means to achieve these goals. Soil microbes may play critical roles in mediating plant productivity and soil C/N dynamics under future climate scenarios of elevated CO 2 (eCO 2 ) through optimizing functioning of the root-soil interface. By using a labeling technique with 13 C and 15 N, we examined the effects of plant growth-promoting Pseudomonas fluorescens on C and N cycling in the rhizosphere of a common grass species under eCO 2 . These microbial inoculants were shown to increase plant productivity. Although strong competition for N between the plant and soil microbes was observed, the plant can increase its capacity to store more biomass C per unit of N under P. fluorescens addition. Unlike eCO 2 effects, P. fluorescens inoculants did not change mass-specific microbial respiration and accelerate soil decomposition related to N cycling, suggesting these microbial inoculants mitigated positive feedbacks of soil microbial decomposition to eCO 2 . The potential to mitigate climate change by optimizing soil microbial functioning by plant growth-promoting Pseudomonas fluorescens is a prospect for ecosystem management.",
"discussion": "Discussion The capability of plants and soil microbes to successfully sequester atmospheric C in terrestrial ecosystems largely depends on plant productivity along with microbial decomposition and mineralization feedbacks within the rhizosphere 8 10 11 12 . Advancing current efforts to mitigate effects of climate change could minimize harmful effects of elevated CO 2 1 26 . Here, for the first time, we show that addition of a microbial inoculant has the potential to promote plant productivity while mitigating positive feedbacks of microbial decomposition to increased plant C inputs that typically accompany eCO 2 . Our results also demonstrated that P. fluorescens inoculation led to increased plant tissue C:N under eCO 2 , resulting in an increased capacity to store C per unit of N in plant tissue. Therefore, this soil microbial inoculant may be a useful tool to mitigate climate change. The results from this study are consistent with numerous field and growth chamber experiments showing that P. fluorescens inoculants can increase plant production under ambient CO 2 14 15 31 , and show that these largely stimulating effects could be additive when combined with eCO 2 . Although it was impossible to exclude potential fertilization effects on plant available N through bacterial cell addition, the increases in total soil N pool by bacterial cell addition (0.016%) is likely negligible in comparison with the increase in plant growth by an average of 42%. In addition, chemical adjustments to litter C:N may contribute to reduced quality and decomposability of plant litter under eCO 2 9 34 which may reduce soil decomposition rates. In comparison to our previous study, overall plant C:N ratios during the rapid vegetative growth stage were lower than during the reproductive stage 35 . Plant roots play an essential role in regulating acquisition of soil nutrients. Nevertheless, little is known about the relationship of root functional traits with plant N use strategies. Root surface area is generally correlated to plant nutrient uptake rates 36 . P. fluorescens inoculants significantly increased root surface area under eCO 2 ( Table S1 ), suggesting that P. fluorescens inoculants could enhance the potential for plant roots to acquire N under N-limited eCO 2 condition. Moreover, our results demonstrated that root surface area positively correlated with plant C:N ratio across experimental treatments ( Figure 2a ), further indicating that the plants' ability to acquire N from the soil could be influenced by soil N availability. Coupled C and N processes in the rhizosphere play a critical role in maintaining the sustainability of ecosystems 34 37 . Recent studies suggest that plant productivity slows when plant N demand decouples from soil N cycling under climate change or other ecological disturbances 38 39 . In our previous study conducted in the same ecosystem as this present work, we found that eCO 2 increased microbial biomass N immobilization and decreased soil N availability 19 . Likewise, this study revealed that eCO 2 significantly decreased soil enzyme C:N stoichiometry ( Table S1 ), indicating greater microbial demand for soil N under eCO 2 40 . N limitation could ultimately dampen ecosystem C sequestration in terms of the eCO 2 fertilization effect on plant productivity 10 34 . However, P. fluorescens inoculant did not directly affect soil enzyme C:N stoichiometry ( Table S1 ). Moreover, the negative relationship between plant biomass C:N and enzyme C:N ( Figure 2b ) suggests that plants can continue to grow through increase in their capacity to store C per unit of N in response to changes in soil N availability, which is mediated by soil microbial activities. It is well known that plants can alter their N uptake rates to cope with plant physiological and environmental changes 41 42 . By using the 15 N isotopic method, we observed that eCO 2 increased the importance of N mineralized from SOM, indicating that the positive effects of eCO 2 on soil N enzyme activities increased soil N availability ( Figure 3a and Table S1 ). However, P. fluorescens inoculants had no detectable effects on δ 15 N values of plant biomass ( Figure 3a ) or on soil N enzyme activities ( Table S1 ). This suggests that P. fluorescens does not facilitate N mineralization under eCO 2 conditions. We note that N in P. fluorescens cells is mostly in organic form with the δ 15 N value of 4.2‰ (the δ 15 N value of organic N in SOM is 587.5‰). If plants took up mineralized N from dead P. fluorescens cells, the δ 15 N value of plant biomass should be lower than plant uptake. However, even if all bacterial N was absorbed by plants, the N in P. fluorescens cells would only contribute from 0.8% to 2.2% of the total plant N pool. In addition, the δ 15 N value of organic N corrected by bacterial cells (587.4‰) was still much higher than inorganic N (445.7‰). eCO 2 -induced rhizosphere priming effects and subsequent microbial N mineralization could influence the magnitude of plant growth 8 21 . Previous studies have identified several plant and microbial traits related to RPE 8 21 43 , but the direct evidence of priming-related effects on plant N availability has not been well documented. Our results, for the first time, clearly demonstrated that priming of SOM decomposition was positively related to plant N availability ( Figure 3b ), suggesting that priming made soil N more available to the plant for uptake. Increased root length was also observed in this study as an important root functional trait related to plant N uptake adaptations associated with microbial plant growth-promoting properties under eCO 2 conditions. These results add to a growing body of evidence that plants could increase N availability through rhizosphere priming and development of root systems to alleviate nitrogen limitation under eCO 2 8 9 21 43 . P. fluorescens inoculants and eCO 2 were expected to increase plant C inputs to soil ( Figure 4a ). However, a synergistic effect of bacteria and eCO 2 on plant-derived C was not observed ( Figure 4a ). This may be due to the use of planting pots which may have constrained root growth in this experiment. Results from a meta-analysis suggest that CO 2 -induced increases in belowground biomass are stronger in plants grown in open fields relative to closed pots 9 . In spite of higher rates of new C inputs, P. fluorescens inoculants and eCO 2 demonstrated contrasting effects on heterotrophic respiration due to microbial activities. eCO 2 increased mass-specific microbial respiration ( Figure 4b ) 35 , consistent with previous observations of climate-induced positive feedbacks 12 40 . P. fluorescens inoculants did not change mass-specific microbial respiration under ambient CO 2 but mitigated positive microbial feedbacks under eCO 2 conditions ( Figure 4b ). Thus, these findings suggest that P. fluorescens inoculants may potentially decrease soil C losses via heterotrophic respiration. Our results indicate that P. fluorescens inoculants may optimize soil microbial functioning and potentially be implemented as a strategy for increasing plant productivity while mitigating positive feedbacks of microbial decomposition to eCO 2 . If the benefits of P. fluorescens inoculants can be scaled from the growth chamber and applied in natural ecosystems in a high-CO 2 world, the potential for terrestrial C sequestration may increase to mitigate rising atmospheric CO 2 . Further assessment is needed to extend these findings to field experiments and to formulate economical methods of inoculation for field deployment. Additional experiments should be performed to assess rhizosphere colonization by P. fluorescens inoculants across a range of plant species."
} | 2,277 |
25784647 | PMC4363858 | pmc | 6,289 | {
"abstract": "Increased plant productivity and decreased microbial respiratory C loss can potentially mitigate increasing atmospheric CO 2 , but we currently lack effective means to achieve these goals. Soil microbes may play critical roles in mediating plant productivity and soil C/N dynamics under future climate scenarios of elevated CO 2 (eCO 2 ) through optimizing functioning of the root-soil interface. By using a labeling technique with 13 C and 15 N, we examined the effects of plant growth-promoting Pseudomonas fluorescens on C and N cycling in the rhizosphere of a common grass species under eCO 2 . These microbial inoculants were shown to increase plant productivity. Although strong competition for N between the plant and soil microbes was observed, the plant can increase its capacity to store more biomass C per unit of N under P. fluorescens addition. Unlike eCO 2 effects, P. fluorescens inoculants did not change mass-specific microbial respiration and accelerate soil decomposition related to N cycling, suggesting these microbial inoculants mitigated positive feedbacks of soil microbial decomposition to eCO 2 . The potential to mitigate climate change by optimizing soil microbial functioning by plant growth-promoting Pseudomonas fluorescens is a prospect for ecosystem management.",
"discussion": "Discussion The capability of plants and soil microbes to successfully sequester atmospheric C in terrestrial ecosystems largely depends on plant productivity along with microbial decomposition and mineralization feedbacks within the rhizosphere 8 10 11 12 . Advancing current efforts to mitigate effects of climate change could minimize harmful effects of elevated CO 2 1 26 . Here, for the first time, we show that addition of a microbial inoculant has the potential to promote plant productivity while mitigating positive feedbacks of microbial decomposition to increased plant C inputs that typically accompany eCO 2 . Our results also demonstrated that P. fluorescens inoculation led to increased plant tissue C:N under eCO 2 , resulting in an increased capacity to store C per unit of N in plant tissue. Therefore, this soil microbial inoculant may be a useful tool to mitigate climate change. The results from this study are consistent with numerous field and growth chamber experiments showing that P. fluorescens inoculants can increase plant production under ambient CO 2 14 15 31 , and show that these largely stimulating effects could be additive when combined with eCO 2 . Although it was impossible to exclude potential fertilization effects on plant available N through bacterial cell addition, the increases in total soil N pool by bacterial cell addition (0.016%) is likely negligible in comparison with the increase in plant growth by an average of 42%. In addition, chemical adjustments to litter C:N may contribute to reduced quality and decomposability of plant litter under eCO 2 9 34 which may reduce soil decomposition rates. In comparison to our previous study, overall plant C:N ratios during the rapid vegetative growth stage were lower than during the reproductive stage 35 . Plant roots play an essential role in regulating acquisition of soil nutrients. Nevertheless, little is known about the relationship of root functional traits with plant N use strategies. Root surface area is generally correlated to plant nutrient uptake rates 36 . P. fluorescens inoculants significantly increased root surface area under eCO 2 ( Table S1 ), suggesting that P. fluorescens inoculants could enhance the potential for plant roots to acquire N under N-limited eCO 2 condition. Moreover, our results demonstrated that root surface area positively correlated with plant C:N ratio across experimental treatments ( Figure 2a ), further indicating that the plants' ability to acquire N from the soil could be influenced by soil N availability. Coupled C and N processes in the rhizosphere play a critical role in maintaining the sustainability of ecosystems 34 37 . Recent studies suggest that plant productivity slows when plant N demand decouples from soil N cycling under climate change or other ecological disturbances 38 39 . In our previous study conducted in the same ecosystem as this present work, we found that eCO 2 increased microbial biomass N immobilization and decreased soil N availability 19 . Likewise, this study revealed that eCO 2 significantly decreased soil enzyme C:N stoichiometry ( Table S1 ), indicating greater microbial demand for soil N under eCO 2 40 . N limitation could ultimately dampen ecosystem C sequestration in terms of the eCO 2 fertilization effect on plant productivity 10 34 . However, P. fluorescens inoculant did not directly affect soil enzyme C:N stoichiometry ( Table S1 ). Moreover, the negative relationship between plant biomass C:N and enzyme C:N ( Figure 2b ) suggests that plants can continue to grow through increase in their capacity to store C per unit of N in response to changes in soil N availability, which is mediated by soil microbial activities. It is well known that plants can alter their N uptake rates to cope with plant physiological and environmental changes 41 42 . By using the 15 N isotopic method, we observed that eCO 2 increased the importance of N mineralized from SOM, indicating that the positive effects of eCO 2 on soil N enzyme activities increased soil N availability ( Figure 3a and Table S1 ). However, P. fluorescens inoculants had no detectable effects on δ 15 N values of plant biomass ( Figure 3a ) or on soil N enzyme activities ( Table S1 ). This suggests that P. fluorescens does not facilitate N mineralization under eCO 2 conditions. We note that N in P. fluorescens cells is mostly in organic form with the δ 15 N value of 4.2‰ (the δ 15 N value of organic N in SOM is 587.5‰). If plants took up mineralized N from dead P. fluorescens cells, the δ 15 N value of plant biomass should be lower than plant uptake. However, even if all bacterial N was absorbed by plants, the N in P. fluorescens cells would only contribute from 0.8% to 2.2% of the total plant N pool. In addition, the δ 15 N value of organic N corrected by bacterial cells (587.4‰) was still much higher than inorganic N (445.7‰). eCO 2 -induced rhizosphere priming effects and subsequent microbial N mineralization could influence the magnitude of plant growth 8 21 . Previous studies have identified several plant and microbial traits related to RPE 8 21 43 , but the direct evidence of priming-related effects on plant N availability has not been well documented. Our results, for the first time, clearly demonstrated that priming of SOM decomposition was positively related to plant N availability ( Figure 3b ), suggesting that priming made soil N more available to the plant for uptake. Increased root length was also observed in this study as an important root functional trait related to plant N uptake adaptations associated with microbial plant growth-promoting properties under eCO 2 conditions. These results add to a growing body of evidence that plants could increase N availability through rhizosphere priming and development of root systems to alleviate nitrogen limitation under eCO 2 8 9 21 43 . P. fluorescens inoculants and eCO 2 were expected to increase plant C inputs to soil ( Figure 4a ). However, a synergistic effect of bacteria and eCO 2 on plant-derived C was not observed ( Figure 4a ). This may be due to the use of planting pots which may have constrained root growth in this experiment. Results from a meta-analysis suggest that CO 2 -induced increases in belowground biomass are stronger in plants grown in open fields relative to closed pots 9 . In spite of higher rates of new C inputs, P. fluorescens inoculants and eCO 2 demonstrated contrasting effects on heterotrophic respiration due to microbial activities. eCO 2 increased mass-specific microbial respiration ( Figure 4b ) 35 , consistent with previous observations of climate-induced positive feedbacks 12 40 . P. fluorescens inoculants did not change mass-specific microbial respiration under ambient CO 2 but mitigated positive microbial feedbacks under eCO 2 conditions ( Figure 4b ). Thus, these findings suggest that P. fluorescens inoculants may potentially decrease soil C losses via heterotrophic respiration. Our results indicate that P. fluorescens inoculants may optimize soil microbial functioning and potentially be implemented as a strategy for increasing plant productivity while mitigating positive feedbacks of microbial decomposition to eCO 2 . If the benefits of P. fluorescens inoculants can be scaled from the growth chamber and applied in natural ecosystems in a high-CO 2 world, the potential for terrestrial C sequestration may increase to mitigate rising atmospheric CO 2 . Further assessment is needed to extend these findings to field experiments and to formulate economical methods of inoculation for field deployment. Additional experiments should be performed to assess rhizosphere colonization by P. fluorescens inoculants across a range of plant species."
} | 2,277 |
25784647 | PMC4363858 | pmc | 6,289 | {
"abstract": "Increased plant productivity and decreased microbial respiratory C loss can potentially mitigate increasing atmospheric CO 2 , but we currently lack effective means to achieve these goals. Soil microbes may play critical roles in mediating plant productivity and soil C/N dynamics under future climate scenarios of elevated CO 2 (eCO 2 ) through optimizing functioning of the root-soil interface. By using a labeling technique with 13 C and 15 N, we examined the effects of plant growth-promoting Pseudomonas fluorescens on C and N cycling in the rhizosphere of a common grass species under eCO 2 . These microbial inoculants were shown to increase plant productivity. Although strong competition for N between the plant and soil microbes was observed, the plant can increase its capacity to store more biomass C per unit of N under P. fluorescens addition. Unlike eCO 2 effects, P. fluorescens inoculants did not change mass-specific microbial respiration and accelerate soil decomposition related to N cycling, suggesting these microbial inoculants mitigated positive feedbacks of soil microbial decomposition to eCO 2 . The potential to mitigate climate change by optimizing soil microbial functioning by plant growth-promoting Pseudomonas fluorescens is a prospect for ecosystem management.",
"discussion": "Discussion The capability of plants and soil microbes to successfully sequester atmospheric C in terrestrial ecosystems largely depends on plant productivity along with microbial decomposition and mineralization feedbacks within the rhizosphere 8 10 11 12 . Advancing current efforts to mitigate effects of climate change could minimize harmful effects of elevated CO 2 1 26 . Here, for the first time, we show that addition of a microbial inoculant has the potential to promote plant productivity while mitigating positive feedbacks of microbial decomposition to increased plant C inputs that typically accompany eCO 2 . Our results also demonstrated that P. fluorescens inoculation led to increased plant tissue C:N under eCO 2 , resulting in an increased capacity to store C per unit of N in plant tissue. Therefore, this soil microbial inoculant may be a useful tool to mitigate climate change. The results from this study are consistent with numerous field and growth chamber experiments showing that P. fluorescens inoculants can increase plant production under ambient CO 2 14 15 31 , and show that these largely stimulating effects could be additive when combined with eCO 2 . Although it was impossible to exclude potential fertilization effects on plant available N through bacterial cell addition, the increases in total soil N pool by bacterial cell addition (0.016%) is likely negligible in comparison with the increase in plant growth by an average of 42%. In addition, chemical adjustments to litter C:N may contribute to reduced quality and decomposability of plant litter under eCO 2 9 34 which may reduce soil decomposition rates. In comparison to our previous study, overall plant C:N ratios during the rapid vegetative growth stage were lower than during the reproductive stage 35 . Plant roots play an essential role in regulating acquisition of soil nutrients. Nevertheless, little is known about the relationship of root functional traits with plant N use strategies. Root surface area is generally correlated to plant nutrient uptake rates 36 . P. fluorescens inoculants significantly increased root surface area under eCO 2 ( Table S1 ), suggesting that P. fluorescens inoculants could enhance the potential for plant roots to acquire N under N-limited eCO 2 condition. Moreover, our results demonstrated that root surface area positively correlated with plant C:N ratio across experimental treatments ( Figure 2a ), further indicating that the plants' ability to acquire N from the soil could be influenced by soil N availability. Coupled C and N processes in the rhizosphere play a critical role in maintaining the sustainability of ecosystems 34 37 . Recent studies suggest that plant productivity slows when plant N demand decouples from soil N cycling under climate change or other ecological disturbances 38 39 . In our previous study conducted in the same ecosystem as this present work, we found that eCO 2 increased microbial biomass N immobilization and decreased soil N availability 19 . Likewise, this study revealed that eCO 2 significantly decreased soil enzyme C:N stoichiometry ( Table S1 ), indicating greater microbial demand for soil N under eCO 2 40 . N limitation could ultimately dampen ecosystem C sequestration in terms of the eCO 2 fertilization effect on plant productivity 10 34 . However, P. fluorescens inoculant did not directly affect soil enzyme C:N stoichiometry ( Table S1 ). Moreover, the negative relationship between plant biomass C:N and enzyme C:N ( Figure 2b ) suggests that plants can continue to grow through increase in their capacity to store C per unit of N in response to changes in soil N availability, which is mediated by soil microbial activities. It is well known that plants can alter their N uptake rates to cope with plant physiological and environmental changes 41 42 . By using the 15 N isotopic method, we observed that eCO 2 increased the importance of N mineralized from SOM, indicating that the positive effects of eCO 2 on soil N enzyme activities increased soil N availability ( Figure 3a and Table S1 ). However, P. fluorescens inoculants had no detectable effects on δ 15 N values of plant biomass ( Figure 3a ) or on soil N enzyme activities ( Table S1 ). This suggests that P. fluorescens does not facilitate N mineralization under eCO 2 conditions. We note that N in P. fluorescens cells is mostly in organic form with the δ 15 N value of 4.2‰ (the δ 15 N value of organic N in SOM is 587.5‰). If plants took up mineralized N from dead P. fluorescens cells, the δ 15 N value of plant biomass should be lower than plant uptake. However, even if all bacterial N was absorbed by plants, the N in P. fluorescens cells would only contribute from 0.8% to 2.2% of the total plant N pool. In addition, the δ 15 N value of organic N corrected by bacterial cells (587.4‰) was still much higher than inorganic N (445.7‰). eCO 2 -induced rhizosphere priming effects and subsequent microbial N mineralization could influence the magnitude of plant growth 8 21 . Previous studies have identified several plant and microbial traits related to RPE 8 21 43 , but the direct evidence of priming-related effects on plant N availability has not been well documented. Our results, for the first time, clearly demonstrated that priming of SOM decomposition was positively related to plant N availability ( Figure 3b ), suggesting that priming made soil N more available to the plant for uptake. Increased root length was also observed in this study as an important root functional trait related to plant N uptake adaptations associated with microbial plant growth-promoting properties under eCO 2 conditions. These results add to a growing body of evidence that plants could increase N availability through rhizosphere priming and development of root systems to alleviate nitrogen limitation under eCO 2 8 9 21 43 . P. fluorescens inoculants and eCO 2 were expected to increase plant C inputs to soil ( Figure 4a ). However, a synergistic effect of bacteria and eCO 2 on plant-derived C was not observed ( Figure 4a ). This may be due to the use of planting pots which may have constrained root growth in this experiment. Results from a meta-analysis suggest that CO 2 -induced increases in belowground biomass are stronger in plants grown in open fields relative to closed pots 9 . In spite of higher rates of new C inputs, P. fluorescens inoculants and eCO 2 demonstrated contrasting effects on heterotrophic respiration due to microbial activities. eCO 2 increased mass-specific microbial respiration ( Figure 4b ) 35 , consistent with previous observations of climate-induced positive feedbacks 12 40 . P. fluorescens inoculants did not change mass-specific microbial respiration under ambient CO 2 but mitigated positive microbial feedbacks under eCO 2 conditions ( Figure 4b ). Thus, these findings suggest that P. fluorescens inoculants may potentially decrease soil C losses via heterotrophic respiration. Our results indicate that P. fluorescens inoculants may optimize soil microbial functioning and potentially be implemented as a strategy for increasing plant productivity while mitigating positive feedbacks of microbial decomposition to eCO 2 . If the benefits of P. fluorescens inoculants can be scaled from the growth chamber and applied in natural ecosystems in a high-CO 2 world, the potential for terrestrial C sequestration may increase to mitigate rising atmospheric CO 2 . Further assessment is needed to extend these findings to field experiments and to formulate economical methods of inoculation for field deployment. Additional experiments should be performed to assess rhizosphere colonization by P. fluorescens inoculants across a range of plant species."
} | 2,277 |
27087107 | null | s2 | 6,291 | {
"abstract": "Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial α/β-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble α-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 Å resolution and revealed a classical α/β-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling."
} | 443 |
34550007 | PMC8550217 | pmc | 6,292 | {
"abstract": "ABSTRACT The ocean represents the largest biome on earth; however, we have only begun to understand the diversity and function of the marine microbial inhabitants and their interactions with macroalgal species. Macroalgae play an integral role in overall ocean biome health and serve both as major primary producers and foundation species in the ecosystem. Previous studies have been limited, focusing on the microbiome of a single algal species or its interaction with selected microbes. This project aimed to understand overall biodiversity of microbial communities associated with five common macroalgal species and to determine the drivers of these communities at ‘Ewa Beach, O‘ahu, HI. Representative species of Chlorophyta (green), Ochrophyta (brown), and Rhodophyta (red) algae, each species having various levels of calcification, thallus complexity, and status as native or invasive species, were collected from an intertidal bench in May 2019. A portion of the V3-V4 variable region of the small-subunit rRNA gene was amplified for high-throughput sequencing using universal bacterial primers to elucidate the core and variable algal microbiome. Significant differences in bacterial community composition were only partially explained by host species, whether the host was native or invasive, and thallus complexity. Macroalgal phylum explained the most variation in associated microbial communities at ‘Ewa Beach. This study advances our understanding of microbial-macroalgal interactions and their connectivity by producing insight into factors that influence the community structure of macroalga-associated microbiota. IMPORTANCE Generally, most eukaryotic organisms form relationships with microbes that are important in mediating host organismal health. Macroalgae are a diverse group of photosynthetic eukaryotic organisms that serve as primary producers and foundational species in many ecosystems. However, little is known about their microbial counterparts across a wide range of macroalgal morphologies, phylogenies, and calcification levels. Thus, to further understand the factors involved in bacterial community composition associated with macroalgal species at one point in time, representative samples were collected across phyla. Here, we show that both host macroalga phyla and morphology influenced the associated microbial community. Additionally, we show that the invasive species Avrainvillea lacerata does not have a unique microbial community on this intertidal bench, further supporting the idea that host phylum strongly influences microbial community composition.",
"introduction": "INTRODUCTION Microorganisms are ubiquitous throughout the environment and form relationships with larger eukaryotic organisms that are important in mediating host health ( 1 , 2 ). Bacterial community composition can be influenced by multiple aspects of the eukaryotic host, such as biogeography ( 3 , 4 ), morphological niche ( 3 , 5 – 9 ), health ( 10 , 11 ), and morphological complexity ( 1 , 6 , 12 – 14 ). These factors can act independently or in association with one another and can vary on an individual level ( 15 ). One group of eukaryotic hosts of interest is macroalgae, a morphologically and taxonomically diverse photosynthetic group of organisms that serve as major primary producers and foundational species within ecosystems ( 16 – 19 ). Additionally, Rhodophyta, Ochrophyta, and Chlorophyta are often found inhabiting the same intertidal and photic zones ( 20 , 21 ). Previous studies have identified specific functions that microbes perform when in association with their hosts ( 22 ). These roles include exchange of nitrogen ( 23 – 25 ), detoxification of pollutants ( 23 , 24 ), the production of secondary metabolites that are directly and indirectly linked to the host functionality ( 11 , 14 , 23 , 26 ), development of host morphology ( 12 ), and the production of essential vitamins such as B 12 ( 27 , 28 ). Marine bacteria organize into biofilms that form a secondary skin on the macroalgal host. This biofilm can influence nutrient uptake and the production of specific chemical cues related to identification and recognition of the host by other flora and fauna ( 19 , 29 – 31 ). In addition to performing certain functions themselves, associated microbiota can encourage specific macroalgal host functions such as signal transduction and gene transfer ( 32 ), growth stimulation ( 1 , 33 , 34 ), morphogenesis ( 12 ), spore germination ( 35 ), nitrogen metabolism ( 1 ), and antifouling defense ( 32 , 36 ). Macroalgal hosts can alter their associated microbiota, selecting for counterparts that are beneficial to their survival ( 29 ). For example, healthy Gracilaria conferta (Rhodophyta) controls its epibiotic colonization through the production of chemical signals ( 32 , 37 ), utilizing the production of specific surface metabolites to attract protective bacteria or deter pathogenic strains ( 29 ). ‘Ewa Beach, O‘ahu, HI, USA, is culturally and historically unique, serving as a collection site of macroalgae, or limu, for local residents. This beach is characterized by a series of rocky intertidal benches interspersed with sand, which provides a hard substrate for macroalgal attachment ( Fig. 1A ). ‘Ewa Beach has historically been impacted by anthropogenic influences, such as nutrient influxes from sewage and sugarcane-based agriculture, that affect long-term ecosystem variations ( 21 , 38 , 39 ). Previous studies have provided descriptions of macroalgal diversity and the processes that influence their structure both spatially and temporally ( 21 , 38 ). Macroalgal diversity at this site had been impacted by both abiotic and biotic factors, specifically, temperature increases and the invasive alga Avrainvillea lacerata (J. Agardh), formerly Avrainvillea amadelpha ( 21 , 40 ). A. lacerata was first identified in Hawaiian subtidal zones in the 1980s ( 41 ) and since then has expanded its range into the intertidal coastal waters ( 21 , 42 ). This species has also been observed in high abundance at mesophotic depths (to 90 m) around western and southern O‘ahu ( 43 ). At ‘Ewa Beach, A. lacerata increased in abundance from <1% cover in 2012 ( 21 ) to 25 to 50% current cover by 2021 in the intertidal waters (H. L. Spalding, personal observations). FIG 1 Map of sample site at ‘Ewa Beach, O‘ahu, HI, USA (A), and morphological identification of macroalgal species in this study: Dictyota sandvicensis (B), Padina sanctae-crucis (C), Asparagopsis taxiformis (D), Halimeda discoidea (E), and Avrainvillea lacerata (F). Bars, 5 cm. To characterize the diversity of macroalgal-associated microbiota, previous research focused on a subset of the microbial community on a single algal genus using culture-dependent techniques ( 14 , 44 – 46 ) or, more recently, using culture-independent analyses of the total microbiota ( 3 , 47 ). In culture-independent analyses, high-throughput DNA amplicon sequencing enables a more comprehensive look at total bacterial community. These types of analyses typically use the highly conserved small-subunit (SSU) rRNA gene because small variations in the gene can indicate large evolutionary distances ( 48 ). The associated analyses typically begin with the construction of operational taxonomic units (OTUs), with SSU rRNA gene sequences clustered based on a 97% similarity threshold, which corresponds to bacterial species level identification ( 49 ). Recently, the divisive amplicon denoising algorithm (DADA2) was developed ( 50 ). DADA2 defines amplicon sequence variants (ASVs) on the basis of error-corrected nucleotide differences and identifies sequence variants from a sample more accurately than OTU-picking algorithms ( 50 , 51 ). In this study, the most abundant species of Chlorophyta, Ochrophyta, and Rhodophyta from ‘Ewa Beach, O‘ahu, HI, were used to examine the microbial-macroalgal diversity. To allow detailed analyses of the host factors as drivers of bacterial community structure, rather than environmental or temporal variations, all samples were collected from the same intertidal bench at the same time. Using culture-independent techniques with ASV identification, this study hypothesized that host phylum influences the microbiota diversity associated with five species of macroalgae. Additionally, because ‘Ewa Beach was recently invaded by A. lacerata , this species was hypothesized to have a distinct microbial community compared to the native macroalgae. Other factors, such as thallus complexity and phylogenetic affinities, were hypothesized to have less influence on the macroalga-associated microbiota diversity.",
"discussion": "DISCUSSION To better understand the microbial diversity of the entire alga under the same environmental constraints, macroalgae were collected at one time point and analyses were completed without separation of tissue specific regions. This showed distinct microbial communities based on host species and thallus characteristics. The microbial communities identified were typical of those associated with marine macroalgal species ( Fig. 2 ) ( 56 – 58 ); however, they may vary based upon environmental conditions which were not measured in this study ( 54 , 59 – 61 ). Abiotic stressors can impact the microbial community structure of multiple species within a single intertidal bench ( 62 ). These distinct relationships were driven by both phylogenetic and functional divisions of the macroalgae and also have been apparent in coral-associated microbial communities of the Great Barrier Reef and the Hawaiian Archipelago ( 63 , 64 ). The presence of Verrucomicrobiota , Actinobacteriota , Bdellovibrionota , Bacteroidota , and Myxococcota in association with abundant macroalgal species at ‘Ewa Beach suggests that these communities may be exposed to anthropogenically altered physicochemical water conditions ( 54 , 55 , 65 – 67 ). Bdellovibrionota has been associated with sewage and sewage-polluted waters, reproducing only in aerobic environments ( 68 ). Potential nitrogen-fixing bacteria present in association with macroalgal assemblages (i.e., Verrucomicrobiota , Alphaproteobacteria , and Cyanobacteria ) increase in response to bioavailable nitrogen ( 69 ). The diversity in energy sources that these bacterial communities utilize may increase the strength of these macroalgal-microbial relationships, especially in response to certain environmental fluctuations ( 70 , 71 ). While drainage pipes have been suggested as a source of nutrients in the ‘Ewa Beach area ( 38 , 64 ), more recent studies found this effect to be limited to areas close to the pipes ( 38 ). The site selected in this study was not adjacent to the drainage pipes examined by Cox and Foster ( 38 ) and was similar in algal species composition to other intertidal environments in ‘Ewa ( 21 ). Additional spatial and temporal sampling is necessary to determine if these bacteria are present in association with macroalgae at other sites in the Hawaiian Archipelago with differing levels of natural (e.g., groundwater) and anthropogenic impacts. Microbial communities can be distinct based on whether a species is native or invasive within a specific environment, further influencing the invasion capacity of the host ( 3 , 45 ). The Executive Summary of the National Invasive Species Management Plan (NISMP) defines the term “invasive species” as “a species that is nonnative to the ecosystem under consideration and whose introduction causes or is likely to cause economic or environmental harm or harm to human health.” However, the microbial community of the invasive A. lacerata was not significantly different from that of H. discoidea (Chlorophyta) or A. taxiformis (Rhodophyta), showing that while host abundance may be influenced by anthropogenic activities, the microbial communities were influenced by the host phylum on this intertidal bench at the time of sampling. Interestingly, A. taxiformis is considered invasive in other ecosystems and possesses a high invasive risk in both tropical and temperate systems through range expansion ( 65 , 66 ). The A. taxiformis samples used in this study may be invasive in origin ( 72 ), which may explain some of the overlap between the Rhodophyta and Chlorophyta communities. Finding no significant difference between microbial counterparts of invasive and native macroalgae supports a stronger influence of phylum on these microbial communities associated with this intertidal bench. However, A. lacerata may be more influenced by the microbial community on the native algae rather than the intrinsic host factors, although the influence of this association under different environmental variables, such as increased water temperatures and nutrient loading, should be examined. Future studies should also include a variety of other native and invasive species replicated spatially across multiple sites. Both Ochrophyta species, P. sanctae-crucis and D. sandvicensis , had microbial community assemblages that were mostly distinct from those of the Rhodophyta and Chlorophyta. Ochrophyta are characterized by their own unique phytochemical profile, strongly attributed to high concentrations of phlorotannins and terpenes ( 73 , 74 ). Phlorotannins play an integral role in ecosystem structure and function, specifically influencing microbial infection ( 73 – 75 ). The majority of identified natural products produced by Ochrophyta are associated with Dictyota spp. ( 76 ). It is therefore likely the production of secondary metabolites by the Ochrophyta that impacts the associated microbiota. Thallus complexity of the host also influenced the associated microbial communities at ‘Ewa Beach. Thallus characteristics are key to the functional role for the macroalgae, and their development may also be directly impacted by associated microbes ( 12 , 77 ). Bacterial communities can experience a functional shift through algal life history ( 78 ), specific host identity, evolutionary history, and morphological complexity ( 13 ); therefore, these differences between hosts were expected. At ‘Ewa Beach, the thallus complexity of the host strongly influenced the community composition of the associated microbiota. These results provide insight into microbial separation based on macroalgal phylum, host species, and thallus complexity. By examining the entire host-associated microbiota from one location at a single time point, this study demonstrates the functional role of macroalgal hosts in influencing their associated microbiota and provides the first description of distinct bacterial communities associated with intertidal macroalgae at one site in ‘Ewa Beach in Hawai’i. Moreover, bacterial communities may influence macroalgal host phylogeny and thallus complexity. Future studies should focus on spatial and temporal comparisons of these macroalgal assemblages to identify the stability of the associated bacterial communities and the influence of anthropogenic nitrogen sources on these assemblages. Furthermore, to determine how microbes impact establishment of invasive algae, additional invasive species should be included in future studies. Collection and analysis of isotope data in future studies may elucidate the drivers of potential nitrogen-fixing bacteria associated with macroalgae. The identification of environmental drivers affecting these relationships, such as temperature and solar irradiance, and the connectivity between ecosystem types in the near future may also reveal connections related to macroalgal and ecosystem health, biodiversity, and overall community composition."
} | 3,918 |
38298685 | PMC10828052 | pmc | 6,293 | {
"abstract": "This study presents an eco-friendly approach for constructing superhydrophobic (S.H.) coatings on steel surfaces. The biо Сu nanoparticles are synthesized using a biоgenic process. Two types of coatings, Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α, were developed and characterized. The EDX results confirm the successful fabrication of two distinct coatings on the steel substrate: one involving the modification of nickel with stearic acid, Ni-Ѕ.Α, and the other involving the modification of nickel with both bio-Cu and stearic acid, Ni-biо Сu-Ѕ.Α. The SEM results revealed that the S.H. coats exhibit circular microstructures which contribute to the surface roughness. The contact angles of water droplets on the Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α coatings were measured at 158° ± 0.9° and 162° ± 1.1°, respectively. Chemical stability tests demonstrated that the Ni-Ѕ.Α coating maintains its S.H. behaviour in a pH range of 3–11, whereas the Ni-biо Сu-Ѕ.Α coating exhibits excellent chemical stability in a broader range of pH (1-13). The coating's mechanical stability was evaluated through abrasion tests. The Ni-Ѕ.Α coating retained its S.H. properties even after an abrasion length equal 1100 mm, while the Ni-biо Сu-Ѕ.Α coating maintained its S.H. behaviour till an abrasion length equal 1900 mm. The corrosion behavior and protective properties of the S.H. coatings were studied via electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization (PDP) techniques. The PDP and EIS findings demonstrated that both Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α coatings significantly reduced the corrosion rate compared to uncoated steel.",
"conclusion": "4 Conclusion In this study, an eco-friendly approach for the construction of two types of superhydrophobic (S.H.) coatings, Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α, was developed. The ԜCA of steel coated with Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α are 158° ± 0.9°, and 162° ± 1.1°, respectively. The chemical stability results obtained in this study reveal that the Ni-Ѕ.Α coating maintains its S.H. characteristics in a pH range of 3–11. On the other hand, the Ni-bio Сu-Ѕ.Α coating demonstrates excellent chemical stability, retaining its S.H. characteristics even in a broader pH range of 1–13. This indicates the robust chemical resistance of these coatings against acidic and alkaline environments. In terms of mechanical stability, the Ni-Ѕ.Α coating exhibits remarkable durability, maintaining its S.H. properties even after an abrasion length of 1100 mm. While, the Ni-biо Сu-Ѕ.Α coating displays exceptional mechanical stability, retaining its S.H. characteristics till an abrasion length of 1900 mm. These findings highlight the strong mechanical integrity and resistance to physical wear of the S.H. coatings. The corrosion behavior and protective properties of S.H. coatings on steel were thoroughly investigated. The corrosion resistance of the coated steel samples was evaluated using potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) techniques. The PDP results revealed that both Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α coatings effectively reduced the corrosion Сurrent density (i corr ) compared to uncoated steel, indicating improved corrosion resistance. The EIS analysis provided valuable insights into the charge transfer resistance (R ct ) and impedance parameters of the coated steel samples. The results showed that the charge transfer resistance of the Ni-Ѕ.Α coated steel was higher than that of uncoated steel, indicating enhanced corrosion resistance. Notably, the Ni-biо Сu-Ѕ.Α-coated steel exhibited the highest R ct value, indicating the most effective barrier against corrosive species. This can be attributed to the presence of biо Сu, which enhanced the superhydrophobicity and formed a protective barrier against corrosive agents.",
"introduction": "1 Introduction Superhydrophobic (S.H.) surfaces, have a water contact angle higher than 150 o and exceptional water resistance, have garnered significant interest for their diverse applications in fields such as drag reduction, sensors, anti-icing, self-cleaning, antifouling, oil-water separation, and corrosion resistance [ [1] , [2] , [3] , [4] , [5] , [6] , [7] ]. Various techniques, including sol-gel, electrospinning, anodization, spraying, electrodeposition, 3D printing, chemical etching, and chemical vapor deposition, have been employed to achieve S.H. surfaces [ [8] , [9] , [10] , [11] , [12] , [13] , [14] ]. Electrodeposition, in particular, stands out as a simple, cost-effective, and flexible method for producing artificial S.H. surfaces [ 14 ]. Despite its advantages, the chemical and mechanical stability limitations, as well as the brittleness of microscopic nanostructures, have hindered the widespread commercialization of S.H. surfaces. To overcome these challenges, researchers have focused on enhancing the chemical stability and mechanical abrasion resistance of S.H. surfaces. One key requirement for achieving S.H. surfaces is to increase surface roughness while simultaneously reducing surface energy [ 15 ]. In the past, perfluorinated compounds were commonly utilized to reduce surface energy due to their extremely low surface energy values [ 14 ]. However, these compounds were associated with toxicity and harmful environmental effects. Consequently, there is a growing need for eco-friendly techniques and materials to produce S.H. surfaces. Researchers have explored various nanomaterials, including metal-organic frameworks, carbon nanotubes, SiO 2 , TiO 2 , and ZnO to enhance surface roughness [ 7 , [16] , [17] , [18] ]. The synthesis of nanoparticles can be achieved through bottom-up (chemical and biоlogical) and top-down (physical) methods [ 19 ]. However, chemical and physical procedures are often environmentally hazardous, toxic, and expensive. Various biоtic resources such as microorganisms, fungi, algae, plants, and actinomycetes have been utilized in the eco-friendly synthesis of nanoparticles [ 20 ]. Plant extracts, in particular, have emerged as potential precursors for the eco-friendly manufacturing of nanomaterials. Active compounds present in plant extracts, including enzymes, carbohydrates, polyphenols, phenolics, and proteins, can act as stabilizers, reducing and capping agents for metal ions, facilitating the formation of metal nanoparticles [ 21 ]. Among the diverse range of nanoparticles, copper nanoparticles have gained more interest as their distinctive chemical and physical properties, make them suitable for numerous domestic and commercial applications. These applications include cosmetics, imaging, catalysis, medical and pharmaceutical purposes, environmental, and energy research applications. Copper nanoparticles find wide utility in the preparation of organic-inorganic nano-composites and as materials for gas sensors, and giant magneto-resistance. Furthermore, copper nanoparticles play a crucial role in medicine as antifungal agents, antibacterial agents, and antioxidants [ [22] , [23] , [24] ]. Steel is a widely used material in various industries due to its versatility, durability, and exceptional strength. It serves as a fundamental component in infrastructure, construction, transportation, manufacturing, and many other sectors [ 25 , 26 ]. However, steel is susceptible to corrosion when exposed to moisture, oxygen, and aggressive environments. Corrosion not only compromises the structural integrity of steel but also leads to significant economic losses due to repairs, replacements, and maintenance. Therefore, effective corrosion protection measures are crucial to extend the lifespan of steel structures, enhance their reliability, and minimize the financial and environmental impact of corrosion. By implementing corrosion protection techniques such as coatings, inhibitors, cathodic protection, and proper maintenance practices, the integrity and performance of steel can be preserved, ensuring its continued contribution to various industries. The investigation into corrosion protection for metals and alloys has gained significant attention in recent years [ 27 ]. Various polymers, both thermoplastic and thermoset, including fluoropolymers, polypropylene, polyurethane, epoxy/silane, and polyetherimide, have proven effective as coatings for corrosion prevention [ 28 ]. In their study, M. Kaseem and H. Choe explored the surface coatings characteristics of bioactive elements produced on Tie-6-Ale-4V alloy through plasma electrolytic oxidation (PEO). The incorporation of Mg and Zn into the surface was achieved by PEO treatment using electrolytes containing Zn 2+ and Mg 2+ ions at varying concentrations [ 29 ]. A.S. Gnedenkov et al. introduced a self-healing PEO-based protective layer design of in-situ grown layered double hydroxides loaded with inhibitors on the MA8 magnesium alloy [ 30 ]. A novel approach has been suggested to create composite coatings that provide active corrosion protection for magnesium alloys. This method involves employing the Plasma Electrolytic Oxidation (PEO) technique and assessing the vulnerability of PEO layers to localized pitting formation using specialized electrochemical methods (SVET/SIET) [ 31 ]. A groundbreaking method was introduced by A. S. Gnedenkov et al. to achieve enhanced corrosion protection for magnesium alloys using intelligent, environmentally friendly hybrid coatings containing inhibitors. This innovative approach involves elevating the corrosion resistance of magnesium and its alloys by creating self-healing hybrid coatings through plasma electrolytic oxidation (PEO) treatment. The process includes synthesizing a ceramic-like bioactive coating on the surface and subsequently infusing the resulting porous PEO layer with the corrosion inhibitor 8-hydroxyquinoline (8-HQ) and the bioresorbable polymer polycaprolactone (PCL) in various configurations, aiming to enhance protective properties [ 32 ]. A. S. Gnedenkov et al. developed a new self-healing coating containing polycaprolactone to improve the corrosion resistance of magnesium and its alloys. This was achieved through plasma electrolytic oxidation (PEO) followed by treatment with an organic biocompatible corrosion inhibitor and a bioresorbable polymer, resulting in the formation of protective layers [ 33 ]. Furthermore, plasma electrolyte oxidation coatings exhibit substantial potential for surface alteration in Mg-based biodegradable constituents, allowing for wide adjustments in composition and properties to regulate degradation and attain biocompatibility [ 34 ]. The objective of this research is to fabricate a superhydrophobic coating on steel using biо-Сu. Two rough coatings of nickel, and nickel grafted with biо-Сu (Ni-biо Сu), were electrostatically deposited onto the steel surface. These coatings were then submerged in an ethanolic solution of stearic acid (S.A) to fabricate S.H. coating. To enhance the chemical and mechanical stability of the S.H. coatings, we innovatively employed biо Сu. To the best of our knowledge, this is the first report utilizing bio Cu as an additive to fabricate a S.H. coating on a steel substrate. Stearic acid, an eco-friendly substance and a cost-effective alternative to toxic fluorinated polymers was chosen as the low surface energy compound. The prepared S.H. coatings were evaluated for their wettability, chemical and mechanical stability, and corrosion resistance in a 1.0 M HCl solution. By addressing these research objectives, we aim to contribute to the development of durable, corrosion-resistant, and eco-friendly superhydrophobic coatings on steel, with potential applications in various industries and sectors.",
"discussion": "3 Results and discussion 3.1 EDX results The EDX technique is used to confirm the composition of the examined Biо Сu, steel coated with Ni-Ѕ.Α, and steel coated with Ni-biо Сu-Ѕ.Α. Fig. 2 illustrates the EDX spectra of Biо Сu, steel coated with Ni-Ѕ.Α, and steel coated via Ni-biо Сu-Ѕ.Α. The EDX micrograph of Biо Сu, Fig. 2 a, reveals the presence of peaks corresponding to Сu NPs, indicating the successful synthesis of Biо Сu NPs. The EDX micrograph of steel coated with Ni-Ѕ.Α, Fig. 2 b, exhibits peaks corresponding to Fe, Ni, C, and O. These elements are expected as they are associated with the steel substrate and the Ni-Ѕ.Α coating. The presence of Fe refers to the steel substrate, while Ni represents the Ni component of the coating. The peaks for C and O are likely associated with the organic stearic acid components in the coating material. In the EDX micrograph of steel coated with Ni-biо Сu-Ѕ.Α, Fig. 2 c, the same peaks observed in the steel coated with Ni-Ѕ.Α are present. Additionally, there is an additional peak corresponding to biо Сu. This confirms the successful incorporation of biо Сu into the formed S.H. coating. Fig. 2 EDX micrographs for a) biо Сu, and steel coated by b) Ni-Ѕ.Α, and c) Ni-biо Сu-Ѕ.Α. Fig. 2 3.2 SEM and wettability Fig. 3 displays the SEM micrographs of the coated steel using Ni-biо Сu-Ѕ.Α and Ni-Ѕ.Α. The discussion regarding the SEM results of the S.H. coated steel with Ni-biо Сu-Ѕ.Α and Ni-Ѕ.Α primarily revolves around the disparities in surface roughness and microstructure between the two coatings. Based on the SEM findings, it is observed that the coating formed with Ni-biо Сu-Ѕ.Α, Fig. 3 b, exhibits smaller circular microstructures in comparison with the coating formed with Ni-Ѕ.Α, Fig. 3 a. This discrepancy in microstructure size can be attributed to the inclusion of biо Сu in the Ni-biо Сu-Ѕ.Α coating, which serves as a nucleation center during the electrodeposition process. The presence of biо Сu accelerates the nucleation process, favoring the formation of smaller structures rather than extensive crystal growth. This, in turn, results in increased surface roughness. The reduced microstructure size contributes to the S.H. characteristics of the coating. Smaller structures tend to enhance surface roughness, leading to a higher stability and water-repellent coating. Consequently, the S.H. characteristics of the coating are improved. Fig. 3 SEM images of coated steel with a) Ni-Ѕ.Α, and b) Ni-biо Сu-Ѕ.Α. Fig. 3 To evaluate the wettability of the Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α coatings, the ԜCA and ԜSA measurements were performed. The Ni-Ѕ.Α coating exhibited a ԜCA of 158° ± 0.9° and a ԜSA of 3° ± 0.1°. On the other hand, the Ni-biо Сu-Ѕ.Α coating demonstrated a ԜCA of 162° ± 1.1° and a ԜSA of 1° ± 0.1°. These results indicate that both coatings possess excellent S.H. properties. 3.3 Chemical stability Chemical stability is a crucial condition for S.H. coatings to maintain their effectiveness over time, especially in harsh environments. The relationship between the solution pH and the ԜCAs and ԜSAs of water droplets on the S.H. coatings is illustrated in Fig. 4 . The findings demonstrate that the Ni-Ѕ.Α films, Fig. 4 a, exhibit superhydrophobicity in a pH range of 3–11. Similarly, the Ni-biо Сu-Ѕ.Α films, Fig. 4 b, exhibit superhydrophobicity in a broader pH range of 1–13. In both cases, the ԜCAs are consistently greater than 150°, indicating strong water repellency, while the ԜSAs remain lower than 10°, indicating excellent water sliding properties. These pH ranges highlight the robustness of the coatings in various alkaline and acidic environments. Fig. 4 The variation of the ԜCA and ԜSA of the S.H. coated steel via a) Ni-Ѕ.Α, and b) Ni-biо Сu-Ѕ.Α with the solution pH. Fig. 4 Comparatively, the chemical stability of the S.H. coated steel with Ni-biо Сu-Ѕ.Α is superior to that of the steel coated with Ni-Ѕ.Α. The inclusion of biо Сu in the Ni-biо Сu-Ѕ.Α coating not only enhances the superhydrophobicity of the coating but also provides an additional layer of protection. This added layer contributes to the improved chemical stability of the coating, making it more resistant to degradation and maintaining its S.H. properties over extended periods. Remarkably, the S.H. coated steel with Ni-biо Сu-Ѕ.Α exhibits superior chemical stability when compared to numerous reported values in the literature [ [36] , [37] , [38] , [39] ] [ [36] , [37] , [38] , [39] ] [ [36] , [37] , [38] , [39] ]. This indicates that the coating's performance surpasses existing studies, further emphasizing its effectiveness in harsh solution conditions and its potential for long-term applications. The coating's ability to withstand a wide pH range and outperform previous research findings highlights its potential for practical applications in corrosive or challenging environments. 3.4 Mechanical stability Superhydrophobic (S.H.) surfaces, despite their advantageous properties, often suffer from reduced practical applications due to their mechanical fragility. In some cases, simply touching S.H. surfaces with a finger can cause them to lose their S.H. characteristics and become damaged [ 40 ]. To assess the resistance of the produced S.H. films to mechanical abrasion, abrasion, and sand impact tests were conducted. Fig. 5 illustrates the change in ԜCAs and ԜSAs of the fabricated S.H. films as a function of the length of abrasion. The Ni-Ѕ.Α film, Fig. 5 a, maintains its S.H behaviour till a length of abrasion equals 1100 mm. In contrast, the Ni-biо Сu-Ѕ.Α film, Fig. 5 b, retains its S.H. properties even till a length of abrasion equals 1900 mm. Impressively, the S.H. coated steel with Ni-biо Сu-Ѕ.Α exhibits superior abrasion resistance when compared to numerous reported values [ 25 , 41 ]. This indicates that the coating's ability to withstand mechanical abrasion surpasses previous studies, making it more durable and suitable for practical applications where mechanical stress is a concern. The enhanced mechanical abrasion resistance of the S.H. coated steel using Ni-biо Сu-Ѕ.Α is due to the presence of the biо Сu layer. The biо Сu layer not only enhances the superhydrophobicity of the coating but also provides an additional protective barrier. This additional layer of protection contributes to the durability and abrasion resistance of the coating, allowing it to maintain its S.H. properties even under harsh mechanical conditions. Fig. 5 The variation in ԜCAs and ԜSAs with the abrasion length for S.H. coated steel via a) Ni-Ѕ.Α, and b) Ni-biо Сu-Ѕ.Α. Fig. 5 Furthermore, sand abrasion tests were conducted to determine the mechanical characteristics of the S.H. coatings, as shown in Fig. 6 . The Ni-Ѕ.Α film, Fig. 6 a, retains its S.H. behaviours till 8 cycles of the sand impact, while the Ni-biо Сu-Ѕ.Α film, Fig. 6 b, maintains superhydrophobicity till 14 cycles of the sand impact. Notably, the Ni-biо Сu-Ѕ.Α coating demonstrates sand impact resistance that exceeds several reported values [ 38 , 42 ]. Fig. 6 The relation between the ԜCAs and ԜSAs of S.H. coated steel with a) Ni-Ѕ.Α, and b) Ni-biо Сu-Ѕ.Α with the sand impact cycles. Fig. 6 3.5 Corrosion measurements 3.5.1 PDP results In addition to the microstructural, chemical stability, and mechanical properties of S.H. coatings, the corrosion characteristics of uncoated steel and S.H. coated steel using Ni-Ѕ.Α and Ni-biо Сu-Ѕ.Α have been investigated through the PDP technique. Fig. 7 presents the PDP plots of bare steel and S.H. coated steel in an aqueous solution of 1.0 M HCl. The PDP curves of uncoated steel in 1.0 M HCl show typical Tafel behavior indicating that the dissolution of steel takes place under the charge transfer process. However, in the presence of S.H. coatings, the anodic part of the graph may not exhibit Tafel behavior, unlike the blank. This observation suggests that the S.H. coatings have a significant impact on the anodic corrosion process. The absence of Tafel behavior in the anodic branch of the PDP graph can be attributed to the inhibiting effect of the S.H. coatings. These coatings create a physical barrier between the corrosive environment (1.0 M HCl) and the steel surface, reducing the availability of corrosive species and hindering the corrosion reaction. The S.H. coatings can alter the mass transport of reactants and products at the steel-coating interface, leading to a modified electrochemical behavior. This alteration in the anodic part of the graph could be due to a combination of factors such as reduced access of corrosive ions to the steel surface, changes in the kinetics of the corrosion reaction, or the creation of a protective film on the steel substrate. It's important to note that the cathodic part of the graph still exhibits Tafel behavior because the reduction reactions involved in corrosion are less affected by the presence of the S.H. coatings. Fig. 7 The PDP plots of bare steel and S.H. coated steel. Fig. 7 Table 2 provides the PDP parameters for uncoated steel and S.H. coated steel, including the protection efficiency (% η), corrosion potential (E corr ), and corrosion current density (i corr ). The % η value is determined using equation (1) : (1) % η = [(i o - i) / i o ] × 100 Where i and i o respectively, stand for the corrosion current densities of S.H. coated steel and uncoated steel. The i corr value for steel coated with Ni-Ѕ.Α is smaller than that of uncoated steel due to the S.H. properties of the coating. The presence of air trapped within the microstructures of the S.H. coating reduces the surface area available for contact between the solution and the steel, leading to a faster decrease in the i corr value. Furthermore, by incorporating biо Сu into the Ni-Ѕ.Α coating, the S.H. properties are enhanced, resulting in a higher reduction in the contact area between the medium and the steel. Consequently, the steel coated with Ni-biо Сu-Ѕ.Α exhibits a higher protection efficiency compared to the coated steel with Ni-Ѕ.Α. These findings highlight the potential of S.H. coatings, particularly those with Ni-biо Сu-Ѕ.Α, for corrosion protection applications in aggressive environments. Table 2 The PDP parameters for the uncoated and the S.H. coated steel in 1.0 M HCl solution. Table 2 Deposit -E corr mV β a mV/decade -β c mV/decade i corr μA/cm 2 % η Uncoated steel 514.5 93.3 85. 5 0.3915723 – Steel + Ni-Ѕ.Α 511.3 195.9 111.6 0.0302125 92.3 Steel + Ni-biо Сu-Ѕ.Α 509.5 212.2 181.9 0.0157012 96.0 3.5.2 EIS results Fig. 8 demonstrates the Nyquist plots and Bode plots comparing the corrosion characteristics of bare steel and S.H. coated steel in a 1.0 M HCl solution. The presence of a depressed capacitive semicircle in the Nyquist plots, Fig. 8 a, at high frequencies, suggests that there is an interfacial charge transfer reaction. The capacitive semicircle size in the Nyquist plot is indicative of the corrosion resistance of the steel. A larger semicircle corresponds to a higher impedance, suggesting a greater resistance to charge transfer and, therefore, a lower corrosion rate. This implies that the steel is less sensitive to corrosion in the presence of the S.H. coatings. When the S.H. coatings are applied to the steel surface, they create a barrier between the corrosive environment and the steel substrate. This barrier reduces the exposure of the steel to the corrosive solution, leading to a decline in the corrosion rate. Consequently, the capacitive semicircle in the Nyquist plot becomes larger, indicating an increase in the impedance and enhanced corrosion resistance. The increase in the size of the capacitive semicircle can be due to the hindrance of the charge transfer reactions at the steel-coating interface. The S.H. coatings prevent the penetration of moisture and ions, reducing the availability of corrosive species and creating a more favorable environment for the creation of a protective layer on the steel substrate. This protective film acts as a physical and chemical barrier, further inhibiting the corrosion process. These results imply that the existence of a protective S.H. layer is what accounts for the increased charge transfer resistance of steel coated with Ni-SA. The steel coated with Ni-biо Сu-Ѕ.Α demonstrates the highest level of protection, as evidenced by the largest capacitive semicircle in the Nyquist plot. Bio-Cu is added to the Ni-coating to increase superhydrophobicity, which enables the Ni-bio Cu-coating to effectively stop the diffusion of corrosive species like Cl − and H 2 O into the steel. The superhydrophobic coating applied to the steel surface demonstrates enhanced impedance magnitudes at lower frequencies in the Bode plots when exposed to a 1.0 M HCl solution, as depicted in Fig. 8 b. This observation affirms the superior protective properties of the manufactured superhydrophobic coating over bare steel. The phase angle plot in Fig. 8 c reveals a distinct time constant at intermediate frequencies, indicating the presence of an electrical double layer. This time constant, occurring at moderate frequencies, is attributed to the role of the electrical double layer in the corrosion protection mechanism. Overall, the Bode and phase angle plots collectively highlight the efficacy of the superhydrophobic coating in mitigating corrosion effects on the steel substrate [ 43 , 44 ]. Fig. 8 a) Nyquist, b) Bode, and c) Phase angle plots of uncoated steel, and S.H. coated steel. Fig. 8 To analyze the experimental results and calculate the impedance parameters, the equivalent circuit presented in Fig. 9 was employed, and the Zsimpwin program was used. The equivalent circuit consists of the charge transfer resistance (R ct ), the constant phase element (CPE), and solution resistance (R s ). Table 3 presents the EIS parameters for both uncoated steel and S.H. coated steel. The % η was calculated using equation (2) : (2) % η = [(R ct - Rct o ) / R ct ] × 100 Where R ct and R cto represent the charge transfer resistance of S.H. coated steel and uncoated steel, respectively. The obtained EIS parameters are listed in Table 3 . It is evident that both R ct and % η values follow the trend: uncoated steel < steel + Ni-Ѕ.Α< steel + Ni-biо Сu-Ѕ.Α. The corrosion resistance of the S.H. coated steel using Ni-biо Сu-Ѕ.Α surpasses several previously reported values [ [45] , [46] , [47] ]. The application of S.H. coatings to the steel results in an elevation of R s , signaling greater solution resistance compared to the uncoated steel. The reduction in CPE for the S.H. coated steel signifies a significant decrease in adsorbed corrosive species to the steel surface, indicating improved protection efficiency for the S.H. coated steel. Moreover, the corrosion resistance of the S.H. coated steel with Ni-biо Сu-Ѕ.Α is greater than that of steel coated with Ni-Ѕ.Α. This can be attributed to the fact that biо Сu enhances superhydrophobicity. This slows down the corrosion process and enhances the overall corrosion resistance of the coating. Fig. 9 The equivalent circuit model. Fig. 9 Table 3 The EIS parameters for the uncoated and S.H. coated steel in 1.0 M HCl solution. Table 3 Coat Rs (Ohm.cm 2 ) n 1 CPE x10 −6 (s n Ω −1 cm 2 ) R ct (Ohm.cm 2 ) % η Uncoated steel 0.2 0.78 53.4 12.1 – Steel + Ni-Ѕ.Α 1.1 0.76 36.2 234.2 94.8 Steel + Ni-biо Сu-Ѕ.Α 1.4 0.72 25.2 439.5 97.2 3.6 Mechanism of corrosion resistance performance Bare steel surfaces possess a propensity to adsorb water molecules, rendering them susceptible to corrosion. The presence of chloride ions on uncoated steel surfaces further exacerbates the corrosion vulnerability. In contrast, steel coated with S.H. films introduces a protective paradigm. The micro- and nanostructures of the S.H. coating, enveloped in a hydrophobic material, contribute to its unique corrosion resistance. The intricate morphology of the S.H. coated steel, with its peaks and valleys, facilitates the entrapment of air within the valleys. The hydrophobic nature of the coating ensures that these air-filled interstices persist, creating a passivation barrier between the steel substrate and the corrosive environment. This air barrier becomes a pivotal element in impeding the progression of corrosion. The obstructive effect of the trapped air plays a crucial role in diminishing the impact of aggressive ion species, notably chloride ions, prevalent in corrosive environments. The hindrance created by the entrapped air limits the direct interaction of these corrosive ions with the underlying steel surface. As a result, the superhydrophobic coating acts as a formidable defense mechanism, reducing the likelihood of aggressive ion attacks and mitigating corrosion [ [48] , [49] , [50] ]."
} | 7,095 |
28245360 | null | s2 | 6,294 | {
"abstract": "The extracellular matrix is an environment rich with structural, mechanical, and molecular signals that can impact cell biology. Traditional approaches in hydrogel biomaterial design often rely on modifying the concentration of cross-linking groups to adjust mechanical properties. However, this strategy provides limited capacity to control additional important parameters in 3D cell culture such as microstructure and molecular diffusivity. Here we describe the use of multifunctional hyperbranched polyglycerols (HPGs) to manipulate the mechanical properties of polyethylene glycol (PEG) hydrogels while not altering biomolecule diffusion. This strategy also provides the ability to separately regulate spatial and temporal distribution of biomolecules tethered within the hydrogel. The functionalized HPGs used here can also react through a copper-free click chemistry, allowing for the encapsulation of cells and covalently tethered biomolecules within the hydrogel. Because of the hyperbranched architecture and unique properties of HPGs, their addition into PEG hydrogels affords opportunities to locally alter hydrogel cross-linking density with minimal effects on global network architecture. Additionally, photocoupling chemistry allows micropatterning of bioactive cues within the three-dimensional gel structure. This approach therefore enables us to tailor mechanical and diffusive properties independently while further allowing for local modulation of biomolecular cues to create increasingly complex cell culture microenvironments."
} | 386 |
22954231 | PMC3472919 | pmc | 6,296 | {
"abstract": "The division of labor between castes and the division of labor in workers according to age (temporal polyethism) in social wasps are crucial for maintaining social organization. This study evaluated the division of labor between castes, and the temporal polyethism in workers of Mischocyttarus consimilis Zikán (Hymenoptera: Vespidae). To describe the behavioral repertory of this species, observations were made of 21 colonies, with 100 hours of observations. In order to observe temporal polyethism, each newly emerged wasp was marked with colored dots on the upper area of the thorax. This allowed the observation of behavioral acts performed by each worker from the time of emergence to its death. Through hybrid multidimensional scaling, a clear division between queens and workers could be identified, in which the behaviors of physical dominance and food solicitation characterized the queen caste; while behaviors such as adult—adult trophallaxis, destruction of cells, alarm, foraging for prey, foraging for nectar, and unsuccessful foraging characterized the worker caste. Hybrid multidimensional scaling characterized two groups, with intra—nest activities preferentially accomplished by younger workers, while extra—nest activities such as foraging were executed more frequently by older workers.",
"introduction": "Introduction Eusocial wasps, including some species of Stenogastrinae and all members of Polistinae and Vespinae, are characterized by overlapping adult generations, reproductive division of labor, and cooperative brood care ( Wilson 1971 ; Michener 1974 ). New colonies are established by independent foundation or by swarming. Wasps of the tribes Polistini and Mischocyttarini and some species of Ropalidiini adopt independent foundation, with one (haplometrosis) or more (pleometrosis) females beginning the construction of the nest, while members of the tribe Epiponini begin new colonies by swarming ( Von Ihering 1896 ; Jeanne 1980 ). In wasps with independent foundation, there is no morphological caste differentiation, and the caste of queen or worker is determined mainly by means of aggressive interactions ( Gadagkar 1991 ). The queens, being more aggressive, can ingest more food during trophallaxis ( Jeanne 1972 ; Röseler 1991 ; Spradbery 1991 ), which may cause them to develop their ovaries ( Queller and Strassmann 1989 ). However, the determination of castes can occur during the pre—imaginal phase ( Hunt 1991 ; O'Donnell 1998a ), affecting the larval nutrition and the rate of development of the immatures, and consequently the division of castes ( West-Eberhard 1969 ; Gadagkar et al. 1991 ; O'Donnell 1998a ). Pre—imaginal determination of caste is evident in swarm— founding species with morphologically distinct castes, including some Epiponini and Ropalidiini ( Jeanne 1991a ), and also in some species with independent foundation, such as Ropalidia marginata ( Gadagkar et al. 1991 ), Belonogaster petiolata ( Keeping 2002 ), and other members of Polistes ( O'Donnell 1998a ). Other investigators, for example Brillet et al. ( 1999 ), have suggested that in some wasp species such as P. dominula , the abdominal vibrations produced by the founders may influence the future status of newly emerged wasps, determining which will become workers or future founders. Sound production during the larval feeding of eusocial wasps was observed by Pratte and Jeanne ( 1984 ) and was described as antennal drumming. In recent studies, Jeanne ( 2009 ) and Suryanarayanan et al. ( 2011 ) suggested that the vibrational signals in the nest may affect caste development, by means of biochemical changes and gene expression at the larval stage. After establishing the colony, the queen must maintain her hierarchy. Early studies indicated that maintaining the dominance hierarchy in colonies of less derived wasps was determined by the presence of the queen laying in the nest, caring for the brood, and attacking other females ( Strassmann 1985 ; Jeanne 1972 ). The queens avoid activities with a high risk of predation and high energy cost, such as foraging, which are carried out more frequently by workers ( Strassmann et al. 1984 ; O'Donnell 1998a ). Therefore the behavioral repertory of workers is generally broader, such as in P. dominula ( Pardi 1948 ), Mischocyttarus drewseni ( Jeanne 1972 ), M. cerberus styx ( Giannotti 1999 ), P. versicolor ( Zara and Balestieri 2000 ), and P. canadensis canadensis ( Torres et al. 2009 ). The division of non—reproductive tasks among nestmates, or polyethism, is one of the largest evolutionary advantages that led to the ecological success of the social insects ( Hölldobler and Wilson 1990 ). Dominance interactions in determining reproductive status in the polistine eusocial wasps ( Pardi 1948 ; West-Eberhard 1969 ) can also structure polyethism in several species ( Reeve and Gamboa 1987 ; Jeanne 1991a ; O'Donnell and Jeanne 1995 ; O'Donnell 1998b , 1998c ). In eusocial Hymenoptera, the division of labor between queens and workers usually increases with the size of the colony ( Jeanne 1986 ), and the degree of temporal polyethism varies with species and also seems to be related to colony size ( Wilson 1971 ; Jeanne 1999 ). In colonies of ants, bees, wasps, and termites with thousands to millions of individuals, there is a clear division of tasks, and workers are highly specialized ( Jeanne 1986 , 2003 ; Hölldobler and Wilson 1990 ). Body size and colony composition are better correlated with the behavioral changes of individuals in the transition from intra—nest to extra—nest tasks, such as foraging activities ( Free 1955 ; Cameron 1989 ; Röseler and Van Honk 1990 ). The development of behavioral specialization in the colony may be related to colony expansion, and consequently to the increase in demanding tasks ( Gaustrais et al. 2002 ). Hence, in small colonies with fewer than a hundred individuals, individuals were observed performing a wide variety of tasks ( Traniello 1978 ; Jeanne 1991b ; Karsai and Wenzel 1998 ), which indicates the existence of high behavioral plasticity. Mischocyttarus consimilis Zikán (Hymenoptera: Vespidae) is an independent— founding eusocial Neotropical wasp. This species was formerly restricted to Paraguay, but has recently dispersed through southern Mato Grosso do Sul and western Paraná states in Brazil. Because of this restricted distribution, the species has been the subject of few studies except those by Montagna et al. ( 2009 , 2010 ) and Torres et al. ( 2011 ). In addition, Montagna and coworkers have recently observed and studied the first case of facultative parasitism in the genus Mischocyttarus , the congener M. cerberus. In view of these features, the aim of this study was to investigate the division of labor in colonies of M. consimilis , in order to better understand aspects related to the evolution of social behavior in wasps.",
"discussion": "Discussion A clear division of labor between queens and workers in colonies of M. consimilis was apparent. Queens spent longer periods of time in the nest, devoting their time to activities of dominance hierarchy and oviposition, while workers were engaged more frequently in maintenance activities, as well as the defense and success of the colonies. This species does not exhibit a well—defined temporal polyethism. However, in general, younger workers spend more time in intra—nest activities and older workers perform more extra—nest activities. These two groups may, if necessary, overlap their repertory, demonstrating a typical behavioral plasticity that occurs in this group of less derived social wasps. The behaviors of physical dominance, food solicitation, and oviposition were executed more often by the queens as also observed in colonies of M. c. styx ( Giannotti 1999 ), P. lanio ( Giannotti and Machado 1999 ), and P. versicolor ( Zara and Balestieri 2000 ). According to Oliveira et al. ( 2006 ), dominance interactions and subordination are increased in large colonies and during post— emergence. Similarly to M. consimilis , in the colonies of M. drewseni , the queens perform most of the ovipositions ( Jeanne 1972 ). This differs from P. lanio , in which oviposition was done exclusively by the queens, confirming the condition of functional monogyny in nests ( Giannotti and Machado 1999 ). According to Deleurance ( 1950 ), the presence of empty cells stimulates oviposition in Polistes , and the queen, by maintaining the cells full of her own eggs, prevents the workers from ovipositing ( Brian 1958 ). On the other hand, the behaviors of adult— adult trophallaxis, alarm, foraging for prey, foraging for nectar, and unsuccessful foraging were carried out more frequently by the workers, as also observed by Giannotti ( 1999 ). As West-Eberhard ( 1969 ) described, during trophallaxis between adults, it was possible to detect a difference between the donor's posture and that of the receiver. The alarm behavior occurred significantly more often in workers, showing that this behavior is important for nest defense. Several studies have shown that during this act, the wasp releases certain volatile substances that function as an alarm pheromone, recruiting nestmates and motivating an attack on the source of disturbance ( Ishay 1965 ; Jeanne 1982 ; Ono et al. 2003 ; Fortunato et al. 2004 ). Concerning the temporal polyethism of M. consimilis , intra—nest tasks such as caring for the offspring are more frequently carried out by younger workers. The high—risk tasks such as foraging and defense of the nest are done by older workers, as occurs in several species of social wasps ( West-Eberhard 1996 ). This division of tasks between older and younger workers occurs through genetic predetermination ( Page and Robinson 1990 ; O'Donnell 1996 ), and according to the conditions of the colony such as the size and age of the offspring, damage to the nest, the presence of predators and parasites, and the size and age of the worker population ( Wilson 1971 ; O'Donnell and Jeanne 1992 ; Inoue et al. 1996 ; Naug and Gadagkar 1998 ). On the other hand, this pattern, as previously described for M. mastigophorus , can be affected by queen—worker and worker—worker interactions ( O'Donnell 1998c ). The queens of species that show independent foundation act as the main precursors, behaviorally regulating the tasks to be accomplished by the workers ( Reeve and Gamboa 1987 ; Gamboa et al. 1990 ). However, in some species, the dominance interaction among workers can induce foraging activity by other workers ( Premnath et al. 1995 ; O'Donnell 1998a ). Therefore, the dominance behavior among workers can play a role in structuring polyethism, even though these workers have little effect on the reproductive competition of the colony ( O'Donnell 1998b ). Dominance interactions in M. consimilis do not seem to have a direct correlation with the frequency of foraging activity as suggested by Premnath et al. ( 1995 ) and O'Donnell ( 1998a ) or with the structuring of polyethism ( O'Donnell 1998b ), since the colonies have fewer workers that must accomplish different tasks from the very first days of life, such as foraging activity, which can be performed in the first week after emergence. The colonies of M. consimilis do not exhibit a well—defined temporal polyethism because most of their activities are carried out throughout their entire lifespans. This pattern evidences a behavioral plasticity among the workers similar to that which occurs in colonies of P. versicolor ( Zara and Balestieri 2000 ). Several studies have shown that independent—founding species have a weak or nonexistent correlation between age and tasks performed by workers ( Cameron 1989 ; Jeanne 1991a ; Giray et al. 2005 ). This characteristic seems to be beneficial to the survival of Mischocyttarus as much as Polistes , because both genera include species with small colonies and with independent foundation ( Giannotti 1999 ; Giannotti and Machado 1994 ). Factors such as the body size and colony composition show better correlations with the behavioral changes of individuals in the transition from intra—nest tasks to outdoor tasks such as foraging activity ( Brian 1952 ; Free 1955 ; Cameron 1989 ; Röseler and Van Honk 1990 ). According to Jeanne et al. ( 1988 ), Polybia occidentalis , a swarm— founding wasp with large colonies, shows a clearer division of tasks according to age. Therefore, the presence of a larger number of workers in a colony allows a well—defined temporal polyethism ( Jeanne et al. 1988 ; O'Donnell 2001 ). However, even in some ‘less derived’ species like a R. marginata ( Naug and Gadagkar 1998 ), there may be a well—defined temporal polyethism. Figure 1. Differences in the relative frequency of records of each behavior between queens and workers in 21 colonies of Mischocyttarus consimilis. Each dot represents a colony. Positive values (right of the vertical line) indicate higher frequency of the behavior in queens, and negative values (left of the vertical line) indicate higher frequency of the behavior in workers. High quality figures are available online. Figure 2. Analysis of division of labor between queen and worker castes by means of ordination by hybrid multidimensional scaling, in two dimensions (stress = 0.25), of the nests of Mischocyttarus consimilis (21 records for queens, filled circles; 21 records for workers, empty circles) according to the 30 defined behavioral acts. The vectors represent the behavioral acts that contributed most to the ordination (r > 0.5). Cl = physical dominance, C8 = food solicitation characterized the repertory of queens, C4 = adult—adult trophallaxis, C14 = destruction of cells, C22 = alarm, C26 = foraging for prey, C27 = foraging for nectar, C30 = unsuccessful foraging characterized the workers' repertory, C23 = immobility was common to both castes. High quality figures are available online. Figure 3. Analysis of temporal polyethism of workers of Mischocyttarus consimilis in five colonies, by ordination by hybrid multidimensional scaling in two dimensions (stress = 0.27). In A the vectors represent the relative contribution of each behavior to the plan of the ordination seen in B (r > 0.5). In B, the size of the points is directly proportional to the workers' age. C4 = adult-adult trophallaxis, C5 = inspection of cells, C6 = chewing prey and feeding larvae, C7 = licking newly emerged, C12 = rubbing the gaster on the cells, C13 = cleaning of cells, C14 = destruction of cells, C16 = nectar storage in the cells, C19 = rubbing the gaster on the petiole, C20 = licking the nest petiole, C23 = immobility, C25 = patrols in the nest, C26 = foraging for prey. High quality figures are available online."
} | 3,700 |
32161149 | PMC7067595 | pmc | 6,297 | {
"abstract": "Jason M. Peters works in the fields of antibiotic resistance and biofuel production. In this mSphere of Influence article, he reflects on how the paper “A global genetic interaction network maps a wiring diagram of cellular function” by Costanzo et al. (Science 353:aaf1420, 2016, https://doi.org/10.1126/science.aaf1420 ) has impacted his work by highlighting the power of gene networks to uncover new biology."
} | 103 |
23894306 | PMC3716776 | pmc | 6,299 | {
"abstract": "Thermophilic bacteria are a potential source of enzymes for the deconstruction of lignocellulosic biomass. However, the complement of proteins used to deconstruct biomass and the specific roles of different microbial groups in thermophilic biomass deconstruction are not well-explored. Here we report on the metagenomic and proteogenomic analyses of a compost-derived bacterial consortium adapted to switchgrass at elevated temperature with high levels of glycoside hydrolase activities. Near-complete genomes were reconstructed for the most abundant populations, which included composite genomes for populations closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus , and for novel populations that are related to thermophilic Paenibacilli and an uncultivated subdivision of the little-studied Gemmatimonadetes phylum. Partial genomes were also reconstructed for a number of lower abundance thermophilic Chloroflexi populations. Identification of genes for lignocellulose processing and metabolic reconstructions suggested Rhodothermus , Paenibacillus and Gemmatimonadetes as key groups for deconstructing biomass, and Thermus as a group that may primarily metabolize low molecular weight compounds. Mass spectrometry-based proteomic analysis of the consortium was used to identify >3000 proteins in fractionated samples from the cultures, and confirmed the importance of Paenibacillus and Gemmatimonadetes to biomass deconstruction. These studies also indicate that there are unexplored proteins with important roles in bacterial lignocellulose deconstruction.",
"introduction": "Introduction Lignocellulosic biomass is an abundant feedstock for the industrial scale production of biofuels as a renewable, carbon-neutral alternative energy source, especially for high energy-density transportation fuels [1] , [2] . The recalcitrance of this biomass to deconstruction into fermentable sugars is a barrier to current biofuel production efforts, and the enzymes required for biochemical deconstruction of biomass are a significant cost in the overall process [3] . Current commercial fungal enzyme cocktails may not be well suited for next-generation biomass pretreatment methods that require elevated temperatures, extreme pH, or those that generate inhibitors or residual pretreatment chemicals such as acids, bases, and/or ionic liquids [4] . Thermophilic microbes may provide a rich alternative source of glycoside hydrolases and other lignocellulolytic enzymes and pathways for biomass deconstruction that can operate efficiently under these environmental conditions [5] – [9] . Enzymes for the deconstruction of lignocellulosic biomass are most often obtained by screening of cultivated microbial isolates, primarily fungi and bacteria [10] . In natural environments, plant biomass is deconstructed by complex microbial communities that employ hydrolytic and oxidative enzymes to depolymerize polysaccharides and lignin [11] . Studying lignocellulose deconstruction by microbial communities, rather than isolates, may provide a more comprehensive view of lignocellulose deconstruction and uncover new microbial groups and deconstruction mechanisms. Natural microbial communities that deconstruct biomass are often complex and it is difficult to assign functional roles to individual microbial groups [12] . For example, microbial communities found in compost that exhibit high rates of biomass deconstruction contain a large number of taxa whose proportions are dynamically altered by changes in substrate composition and temperature [13] . Enrichment cultures established with defined substrates and at constant temperatures offer the possibility of simplifying these complex microbial communities and identifying functional roles for specific populations within the community. The feasibility of targeted discovery of glycoside hydrolases from metagenomic sequencing was demonstrated in a solid state switchgrass-adapted community [7] , [14] . More recently, bacterial consortia have been adapted to switchgrass deconstruction under thermophilic conditions in liquid culture, resulting in low diversity bacterial consortia with a few dominant members and high levels of xylanase and endoglucanase activity [8] . Members of the Firmicutes, Thermi , and Bacteriodetes were the most abundant community members, with members of an uncultivated lineage of the Gemmatimonadetes and thermophilic Chloroflexi present at lower abundances. The supernatants were used to saccharify ionic liquid ([C 2 mim][OAc]) pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that this type of consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to process conditions relevant within the biorefinery context [9] . Shotgun sequencing of microbial consortia (metagenomics) is a powerful method to determine the metabolic potential of multi-species consortia without the bias inherent in microbial cultivation [15] . This method has been applied broadly to study natural and engineered microbial communities [16] . Companion proteomic measurements, referred to as community proteogenomics or metaproteomics, identify which proteins predicted by the metagenomics are produced by the microbial community [17] – [19] . Here we describe the application of proteogenomics to switchgrass-adapted communities to help define the metabolic roles of dominant uncultivated populations in the complex process of biomass deconstruction.",
"discussion": "Discussion Applying proteogenomic methods to a thermophilic bacterial consortium adapted to grow on switchgrass has revealed functional roles for community members and identified proteins that may be involved in switchgrass deconstruction. Analysis of metagenomic sequencing data identified the most abundant populations, as measured by read depth of assembled contigs, as closely related to sequenced strains of Thermus thermophilus and Rhodothermus marinus . Interestingly, the fraction of the proteome assigned to Rhodothermus (8%) was lower than proteins assigned to Paenibacillus (16%) and Gemmatimonadetes (22%), which were at lower relative abundance in the metagenome. This discrepancy may have arisen because the proteome measurements were performed on a later passage of the switchgrass adapted community, and the relative ratios of the abundant populations has been shown to fluctuate in switchgrass-adapted enrichments [60] , but could also reflect differences in relative activity. The Thermus and Rhodothermus populations were closely related to sequenced organisms; however, the sequences binned as Paenibacillus and Gemmatimonadetes were not closely related to sequenced isolates and the reconstructed genomes represent novel composite genomes, with average amino acid identity of 56% and 48% respectively, including a large number of novel carbohydrate or lignin active enzymes. This novelty, combined with the fact that three of the closest reference genomes ( R. marinus , T. terrenum , and S. thermophilus ) were only recently sequenced as part of the Genomic Encyclopaedia of Bacteria and Archaea project [61] , also highlights the need for further sequencing of reference genomes in poorly represented phyla to improve metagenomic analyses. Phylogenetic analysis of the 16S rRNA gene recovered from the Gemmatimonadetes population in the adapted consortium indicates that it is affiliated with subdivision 5 of the phylum (Gemm-5) [8] , the first genomic representation for this subdivision. The composite genome reconstructed for the Gemmatimonadetes population showed that it is substantially divergent from the sole reference isolate and genome for this phylum, Gemmatimonas aurantiaca \n [51] , which belongs to a separate subdivision (subdivision 1), comparable with the divergence of alpha- and gamma-proteobacteria. The majority of Gemmatimonadetes 16S rRNA gene sequences have been recovered from soil, with the highest proportion found in more arid soils [52] . Gemmatimonadetes 16S rRNA gene sequences have been recovered from compost samples; however these sequences were at low abundance [62] . Therefore, the Gemmatimonadetes composite genome recovered from this switchgrass-adapted community represents an unexplored branch of this phylum, as it is enriched under thermophilic conditions in liquid media. Inspection of the Gemmatimonadetes genome revealed that it has multiple glycoside hydrolases responsible for cellulose and hemicellulose hydrolysis as well as a number of multicopper oxidase genes predicted to be laccases, possibly involved in lignin oxidation. The predicted role for Gemmatimonadetes in biomass deconstruction was further supported by subsequent proteomic analysis (see below). In addition to Gemmatimonadetes , the Paenibacillus and Rhodothermus populations were predicted by the metagenome to be involved in biomass deconstruction. The reconstructed Paenibacillus genome has a large number of genes for biomass deconstruction, including a complete set of genes for cellulose hydrolysis (cellobiohydrolase, endoglucanase, β-glucosidase). The presence of a broad complement of glycoside hydrolases in the Paenibacillus population is consistent with genomes of related Gram-positive Firmicutes such as Paenibacillus sp. strain JDR-2 ( http://www.cazy.org/b991.html ). The reconstructed genome for the Rhodothermus population has a very similar gene content and high amino acid identity (96%) to the genome of Rhodothermus marinus , isolated from a submarine alkaline hot spring in Iceland [63] . This thermophile has been shown to secrete endoglucanase, xylanase and alpha-L-arabinofuranosidase enzymes when grown in the presence of biomass substrates [64] – [66] . A remarkable aspect of the comparison between the reconstructed Rhodothermus genome from the switchgrass-adapted consortium and the R. marinus genome is that they are so similar despite being recovered from highly divergent environments. While metagenomic sequences outlined the broad metabolic capabilities of the abundant populations present in the switchgrass adapted community, proteomic data allowed us to focus on the pathways that are actually expressed, and refine the assignment of roles for community members in biomass deconstruction. Proteins for the deconstruction of starch (GH13, GH31) were abundant and had a broad distribution in the proteome. GH13 proteins were detected from Paenibacillus , Rhodothermus , Sphaerobacter and Thermus . This observation is consistent with the presence of water-insoluble starch in the switchgrass, which had been extracted with hot water to remove soluble components [8] . The amorphous starch present, a non-structural component of switchgrass, would be readily accessible to enzymes in comparison to cellulose. The majority of the other glycoside hydrolases detected by proteomics were involved in the deconstruction of hemicellulose. A GH74 family putative xyloglucanase from Sphaerobacter is the most abundant overrepresented glycoside hydrolase in the supernatant, followed by an alpha-L-arabinofuranosidase (GH51) from Paenibacillus , and xylanases (GH10) from Paenibacillus and Hyphomicrobium . This observation is consistent with the high levels of xylanase activity observed in the supernatant of the switchgrass and supports a model in which xylan is the primary polysaccharide that is hydrolyzed by the switchgrass adapted community during growth. In contrast, only one cellulase, an endoglucanase (GH5) from Paenibacillus , was abundant and overrepresented in the supernatant, which mirrored the relatively low level of endoglucanase activity recovered from the switchgrass-adapted community. Comparison of the supernatant proteome of the switchgrass-adapted community with the proteome of the same consortium that had been subsequently perturbed by cultivation with microcrystalline cellulose [9] demonstrated that the complement of glycoside hydrolases was different for the cellulose community. In particular, in the supernatant from the cellulose-perturbed community, a cellobiohydrolase (GH48), two endoglucanases (GH5 and GH9), a xylanase (GH10) and an alpha-L-arabinofuranosidase were detected from Paenibacillus that were not detected in the switchgrass-adapted cultures. These results indicate that bacteria have a differential response to the composition of biomass substrates, a phenomenon which has also been observed in quantitative proteomic analysis of Clostridium thermocellum and Caldicellulosiruptor obsidiansis \n [67] , [68] . A number of unexpected proteins were detected in the supernatant proteome that hint at unexplored steps in lignocellulose deconstruction by bacteria. Sugar isomerases were overrepresented in the supernatant proteome and detected from multiple community members, despite their documented roles in cytoplasmic sugar catabolism. These detections may result from cell lysis during the two week cultivations; however, extracellular xylose isomerases have been found in Streptomyces sp. (strain NCL 82-5-1) [69] and a thermophilic Bacillus sp. (NCIM 59) [70] , and have also been observed at high relative levels in C. obsidiansis grown on acid-treated switchgrass. Therefore the sugar isomerases may have an extracellular role in transforming monosaccharides produced from hemicellulose hydrolysis, potentially as a means of relieving product inhibition on the primary hemicellulases, or as a way to circumvent competition for these monosaccharides from other members of the microbial community. A second set of unexpected proteins in the supernatant proteome were superoxide dismutase and Mn-catalase, two proteins that detoxify oxygen radicals. Though these proteins are often associated with intracellular processes, recent iTRAQ proteomic analyses of the supernatant of lignin-grown Thermobifida fusca demonstrate that both of these proteins were present in the supernatant [71] . The presence of these proteins suggests that oxygen radicals are produced by the microbes in the extracellular medium, and these radicals may be involved in lignin deconstruction. The detection of only one possible lignin deconstructing enzyme in the supernatant proteome, a laccase from Gemmatimonadetes, provides further evidence for our current lack of understanding of bacterial lignin deconstruction. In conclusion, we have used proteogenomics to propose roles for individual community members in the deconstruction of switchgrass by a thermophilic bacterial consortium. From the metagenomics analysis, we were able to reconstruct multiple nearly complete draft genomes, including the genome of an uncultivated lineage in the Gemmatimonadetes . Proteomic analysis indicated the central role played by Paenibacillus in biomass deconstruction and confirmed that hemicellulose hydrolysis was a primary activity of the switchgrass-adapted community."
} | 3,746 |
27434424 | PMC5315479 | pmc | 6,300 | {
"abstract": "Marine oxygen minimum zones (OMZs) are expanding regions of intense nitrogen cycling. Up to half of the nitrogen available for marine organisms is removed from the ocean in these regions. Metagenomic studies have identified an abundant group of sulfur-oxidizing bacteria (SUP05) with the genetic potential for nitrogen cycling and loss in OMZs. However, SUP05 have defied cultivation and their physiology remains untested. We cultured, sequenced and tested the physiology of an isolate from the SUP05 clade. We describe a facultatively anaerobic sulfur-oxidizing chemolithoautotroph that produces nitrite and consumes ammonium under anaerobic conditions. Genetic evidence that closely related strains are abundant at nitrite maxima in OMZs suggests that sulfur-oxidizing chemoautotrophs from the SUP05 clade are a potential source of nitrite, fueling competing nitrogen removal processes in the ocean.",
"conclusion": "Conclusions Data resulting from the cultivation of “ Ca. T. autotrophicus” strain EF1 have expanded the roles of SUP05 in the marine nitrogen cycle. They suggest that SUP05 are a potential source of NO 2 − and sink for NH 4 + in anoxic marine waters. In the conceptual metabolic coupling model of an OMZ proposed by Hawley et al. (2014) , ammonia-oxidizing archaea are the source of NO 2 − in the upper and lower oxycline and SUP05 are the hypothesized source of NH 4 + at and below the lower oxycline. If under the right conditions SUP05 produce NO 2 − , then elevated concentrations of NH 4 + are not required to account for the elevated NO 2 − concentrations at and below the lower oxycline. There is strong evidence indicating that NO 3 − reduction is an independent process that can account for a significant fraction of NO 2 − accumulation in OMZs ( Lam et al. , 2009 ). We hypothesize that SUP05 contribute to the secondary NO 2 − maxima when NO 3 − fluxes are relatively high, ammonia concentrations are relatively low and oxygen concentrations fall below 4 μ M . Our physiological data suggest that SUP05 cells require between 0.03 to 0.06 μ M of NH 4 + for every μ M of NO 2 − produced ( Figures 3 and 4 ). This is in stark contrast to ammonia-oxidation, which is 1:1. It suggests that chemoautotrophic members of the SUP05 clade are an important source of NO 2 − in OMZs where NH 4 + is limiting.",
"introduction": "Introduction Nitrogen is a limiting nutrient in much of the world's ocean. Thirty to fifty percent of the fixed nitrogen available for marine organisms is lost from the ocean due to the biological production of dinitrogen gas (N 2 ) in oxygen minimum zones (OMZs) ( Codispoti et al. , 2001 ; Galloway et al. , 2008 ). Nitrogen loss in these regions is attributed to two microbially mediated processes, heterotrophic denitrification ( Jayakumar et al. , 2004 ; Castro-Gonzalez et al. , 2005 ; Ward et al. , 2009 ; Babbin et al. , 2014 ) and anaerobic ammonia oxidation (anammox) ( Kuypers et al. , 2005 ; Thamdrup et al. , 2006 ; Hamersley et al. , 2007 ; Lam et al. , 2009 ). Genomic data have identified an abundant group of sulfur-oxidizing marine chemoautotrophs (SUP05) that are assumed to contribute to either denitrification or anammox ( Walsh et al. , 2009 ; Canfield et al. , 2010 ; Zaikova et al. , 2010 ; Ulloa et al. , 2012 ; Wright et al. , 2012 ; Mattes et al. , 2013 ; Hawley et al. , 2014 ; Murillo et al. , 2014 ). Members of the SUP05 clade are hypothesized to contribute directly by sequential reduction of nitrate (NO 3 − ) to nitrogenous gases (N 2 O or N 2 ), or indirectly by dissimilative NO 3 − reduction to ammonia (DNRA), which can in turn fuel anammox ( Walsh et al. , 2009 ; Canfield et al. , 2010 ; Hawley et al. , 2014 ; Murillo et al. , 2014 ). SUP05 also have the genetic potential to produce nitrite (NO 2 − ), which is a critical intermediate in denitrification and a necessary reductant for anammox. The sources of NO 2 − , and in particular the secondary NO 2 − maximum, in OMZs are poorly understood ( Lam et al. , 2009 ). Accumulation of NO 2 − in OMZs has been attributed to heterotrophic denitrification leading to N-loss ( Lam et al. , 2009 ). The current paradigm is that NO 2 − in OMZs is produced at the oxycline by aerobic ammonia oxidizing archaea and bacteria (AOA, AOB) ( Hawley et al. , 2014 ) and within the OMZ by heterotrophs ( Codispoti et al. , 2001 ; Francis et al. , 2007 ; Lam et al. , 2009 ). Chemoautotrophic SUP05 also have the genetic potential to respire NO 3 − and produce NO 2 − ( Walsh et al. , 2009 ; Hawley et al. , 2014 ; Murillo et al. , 2014 ). Here we test the hypothesis that a chemoautotrophic bacterium from the SUP05 clade respires NO 3 − and produces NO 2 − . We isolated a representative from the SUP05 clade to elucidate the effects of these sulfur-oxidizing bacteria on the marine nitrogen cycle. We describe the isolation, genetic potential and growth requirements of a sulfur-oxidizing chemolithoautotroph that produces NO 2 − and is limited by ammonium (NH 4 + ). We provide insights into the hypothesized roles of SUP05 in the marine nitrogen cycle that could not be inferred from environmental sequence data alone. Our primary finding suggests that SUP05 produce NO 2 − in OMZs, a critical intermediate required for nitrogen removal processes (anammox and denitrification). We propose the following name for the first isolate from the SUP05 clade: Thioglobus gen. ( Marshall and Morris, 2013 ). “ Candidatus Thioglobus autotrophicus” sp. nov Etymology: au.to.tro'phi.cus. Gr. n. autos self; Gr. adj. trophikos nursing, tending or feeding; N.L. masc. adj. autotrophicus autotroph.",
"discussion": "Results and Discussion Isolation of a representative from the SUP05 clade Six out of 192 culture wells inoculated with water from a redox gradient in Effingham Inlet were positive for growth after 21 days. Four of the cultures had identical 16S rRNA gene sequences and were identified as members of the SUP05 clade. One culture was identified as a closely related gamma-proteobacterium just outside the clade and one culture was identified as an epsilon-proteobacterium related to Arcobacter sp. associated with marine sponges. All are suspected sulfur-oxidizing bacteria. “ Ca. T. autotrophicus” strain EF1 was selected for further study. The remaining cultures were cryopreserved. Phylogenetic analysis and genome sequencing further confirmed the identity and genetic potential of “Ca. T. autotrophicus” strain EF1 ( Figure 1 ). Strain EF1 is most closely related to sequences from the original SUP05 clade described by Walsh et al. (2009) . These include environmental clones recovered from a broad range of OMZs and symbionts of deep-sea mollusk Bathymodiolus sp. Related sequences derived from the whole genomes of symbionts, and from environmental clones from the northeast Pacific ridge ( Huber et al. , 2006 ), the Namibian upwelling system ( Lavik et al. , 2009 ), Suiyo Seamount ( Sunamara et al. , 2004) , Saanich Inlet ( Walsh et al. , 2009 ), the Eastern North Pacific and Eastern South Pacific ( Stevens and Ulloa, 2008 ), the South Atlantic and North Pacific Gyres ( Swan et al. , 2011 ) and Puget Sound ( Marshall and Morris, 2013 ). The complete 16S rRNA gene sequences obtained from the “ Ca . T. singularis” strain PS1 and “ Ca . T. autotrophicus” strain EF1 genomes were also analyzed using the SILVA high quality ribosomal RNA database ( Quast et al. , 2013 ). “ Ca . T. singularis” strain PS1 was most closely related to sequences in the Arctic96BD-19 subclade (ZD0405 in SILVA). “ Ca . T. autotrophicus” strain EF1 was most closely related to sequences in the SUP05 subclade. The purity of “ Ca. T. autotrophicus” strain EF1 was confirmed by several methods. TRFLP analyses identified a single 266 bp fragment using the restriction enzyme MboI and a 193 bp fragment using the restriction enzyme HaeIII ( Supplementary Figure 2 ). These exactly match the fragments predicted from the 16S rRNA gene sequence. No other fragments were observed. Purity was further confirmed by quantitative fluorescence in situ hybridization (FISH) analyses with a SUP05 specific probe (GSO-1032) that exactly matches the 16S rRNA ( Glaubitz et al. , 2013 ) ( Supplementary Figure 2 ). All of the DAPI stained objects observed in three images (117/115/118) also hybridized to the SUP05 probe (total=350/350). Cultures were subsequently cryopreserved and revived from glycerol stocks several times and under both aerobic and anaerobic growth conditions. Sequence and restriction analyses were used to check purity every time a culture was revived from glycerol stocks and before and after every physiology experiment. In every case, these analyses produced the same 16S rRNA gene sequences and the same restriction patterns. Transmission electron microscopy images revealed a single morphology, indicating that strain EF1 is a small (~0.3–0.4 μm) cocci shaped bacterium that produces extracellular globules resembling those produced by “ Ca . T. singularis” PS1 ( Supplementary Figure 3 ). Genetic potential of “Ca. T. autotrophicus” strain EF1 The complete genome of “ Ca. T. autotrophicus” strain EF1 has a GC content of 39.1%. It codes for 1,506 proteins, 92 pseudogenes, 3 rRNAs (5S, 16S and 23S) and 35 tRNAs. It has the genetic potential to grow as a facultatively anaerobic chemolithoautotroph that oxidizes sulfur and can reduce O 2 , NO 3 − and NO ( Figure 1b ). The genome codes for key enzymes for carbon fixation via the Calvin-Benson-Bassham (CBB) cycle, including cbbYCOQR, carbonic anhydrase and cytochrome cbb 3 , one copy of the small subunit of RuBisCO (form I) and two copies of the large subunit of RuBisCO (form I and form II) ( Badger and Bek, 2008 ). RuBisCO form I is composed of large and small subunits and is present in most chemoautotrophic bacteria, cyanobacteria, red and brown algae and all plants. Form II is a dimer of large subunits and is present in purple non-sulfur bacteria, some chemoautotrophic bacteria and in dinoflagellates. Some non-sulfur phototrophic bacteria contain both forms of RuBisCO. Strain EF1 is facultatively anaerobic and has likely adapted to use form IA RuBisCO or form II RuBisCO, depending on the ratio of CO 2 to O 2 . Strain EF1 codes for complete glycolytic and phosphogluconate pathways (nonoxidative), and has most genes encoding the tricarboxylic acid (TCA) cycle. “ Ca. T. autotrophicus” strain EF1 does not code for α-ketoglutarate dehydrogenase, a key TCA enzyme that is also missing from closely related symbiont genomes and a planktonic SUP05 population genome from Saanich Inlet ( Walsh et al. , 2009 ). The absence of α-ketoglutarate dehydrogenase suggests that “ Ca. T. autotrophicus” strain EF1 is an obligate autotroph ( Wood et al. , 2004 ). Cytochrome c oxidase was also identified, along with a suite of genes for oxidative phosphorylation, further indicating the potential for strain EF1 to grow under aerobic conditions. Genes for chemoautotrophic energy conservation were also identified. These include genes for inorganic sulfur oxidation ( fccAB, dsrABCH, aprABM, soxABXYZ and rhodanese sulfurtransferase ) and for aerobic and anaerobic respiration on O 2 , NO 3 − and NO ( narQGHIJ, napABGD and norBCD ) ( Figure 1b ). Sulfur oxidation genes confer the ability to oxidize a broad range of reduced sulfur compounds, including hydrogen sulfide (H 2 S), elemental sulfur (S 0 ) and thiosulfate (S 2 O 3 2− ). Similar to Saanich Inlet SUP05, strain EF1 is also missing soxCD sulfur dehydrogenase genes, suggesting that they store S 0 . The absence of soxCD has coincided with the ability of a closely related symbiont, Ruthia magnifica, to store sulfur in the form of extracellular globules ( Newton et al. , 2007 ). Genes for anaerobic respiration confer the ability to carry out two of the four steps associated with sequential denitrification (NO 3 − → NO 2 − → NO → N 2 O → N 2 ). Strain EF1 has the genetic potential to reduce nitrate to nitrite (NO 3 − → NO 2 − ) and to reduce nitric oxide to nitrous oxide (NO → N 2 O) ( Figure 1b ), but lacks genes to reduce nitrite to nitric oxide (NO 2 − → NO) or to reduce nitrous oxide to nitrogen gas (N 2 O → N 2 ). Although the ability to use NO 2 − and N 2 O as terminal electron acceptors has been observed in environmental datasets ( Walsh et al. , 2009 ; Hawley et al. , 2014 ; Murillo et al. , 2014 ). We also found that strain EF1 is missing key genes required to use hydrogen gas (H2) as an electron donor, as previously reported for environmental SUP05 in the Guaymas Basin ( Anantharaman et al. , 2013 ) “ Ca . T. autotrophicus” strain EF1 has key genes required to use either NH 4 + or organic nitrogen for biosynthesis ( Figure 1b ). These include genes that confer the ability to regulate intracellular nitrogen and to assimilate NH 4 + and amino acids. Strain EF1 codes for two ammonia transporters, NADPH-dependent glutamate synthase (GS) and glutamine oxoglutarate aminotransferase (GOGAT), as well as components of amino acid (AA) and peptide ABC-transporters. Growth requirements Physiology experiments confirmed that strain EF1 is a facultatively anaerobic chemolithoautotroph that requires inorganic sulfur as a source of electrons ( Figure 2 ). Batch cultures grew to an average final cell density of 3.6 × 10 6 cells/ml under aerobic conditions in seawater media containing 1 m M S 2 O 3 2− and to an average final cell density of 2.8 × 10 6 cells/ml under anaerobic conditions in seawater media amended with 1 m M S 2 O 3 2− , 100 μm of NO 3 − and sparged with a mixture of N 2 :CO 2 ( Figures 2a and b , respectively). Cells were unable to grow in aerobic seawater media that lacked S 2 O 3 2− or in anaerobic media that lacked either S 2 O 3 2− or CO 2 . This indicates that “ Ca . T. autotrophicus” strain EF1 requires a reduced form of inorganic sulfur for electrons and CO 2 for biosynthesis. We have found that strain EF1 cells survive two transfers with no additional sulfur ( Supplementary Figure 3 ). This is likely due to their potential to store sulfur in extracellular globules ( Walsh et al. , 2009 ; Marshall and Morris, 2013 ; Hawley et al. , 2014 ). No growth was observed when “ Ca . T. autotrophicus” strain EF1 was grown with H 2 as an electron donor ( Supplementary Figure 4 ). We grew strain EF1 on copiotrophic media ( Supplementary Table 2 ) enriched with NO 3 − to further evaluate its potential for dissimilatory NO 3 − reduction under anaerobic conditions ( Figure 3 ). Cultures grew to similar cell densities at in situ concentrations of NO 3 − (32 μm) and when 100 μm of additional NO 3 − was added to the media ( Figure 3a ). Strain EF1 was not limited by NO 3 − on copiotrophic seawater media. In both cases there was strong evidence for dissimilatory NO 3 − reduction, as indicated by a 1:1 conversion of NO 3 − to NO 2 − ( Figure 3b ), as well as uptake of NH 4 + ( Figure 3c ) and an increase in the production of N 2 O ( Figure 3d ). Although NO was not added to the media, some NO may have been present in the seawater used to make the media or introduced into as a contaminant via the N 2 :CO 2 gas mix used to sparge the media. Regardless, growth experiments support genomic predictions and previously published results from the field indicating that SUP05 produce N 2 O ( Walsh et al. , 2009 ; Hawley et al. , 2014 ). EF1 cells did not produce N 2 gas ( Supplementary Figure 4 ). The potential for “ Ca. T. autotrophicus” strain EF1 to respire NO 3 − and assimilate NH 4 + was evaluated further under nitrogen limitation using oligotrophic seawater media ( Figure 4 , Supplementary Table 2 ). Cultures grew to the highest final cell densities (average 1.2 × 10 6 cells/ml) in media that was amended with 1 m M S 2 O 3 2− , 100 μm NO 3 − and 5 μm NH 4 + ( Figure 4a ). Cells grew to lower cell densities (average 6.2 × 10 5 cells/ml) and had slower growth rates when only S 2 O 3 2− and NO 3 − were added to the media. There was a 1:1 conversion of NO 3 − to NO 2 − in treatments amended with S 2 O 3 2− and NO 3 − , or with S 2 O 3 2− , NO 3 − and NH 4 + ( Figure 4b ). The amount of NO 3 − converted to NO 2 − increased four-fold (average increase from 5 μm to 22 μm) when NH 4 + was added to the media and there was a decrease in NH 4 + concentration over time ( Figure 4b ). Some growth was observed in S 2 O 3 2− only controls. This is likely due to the low concentrations of NO 3 − (0.15 μ M ) and NH 4 + (0.04 μm) present in oligotrophic seawater media ( Supplementary Table 2 ). This experiment further confirmed that “ Ca. T. autotrophicus” strain EF1 was unable to use NO 2 − as a terminal electron acceptor ( Figures 4c and d ). When cells were amended with NO 2 − instead of NO 3 − , no difference in growth was observed between amendments and controls. NO 2 − and NH 4 + concentrations remained constant throughout the experiment. The potential to assimilate amino acids (Promega, Madison, WI, USA, Amino Acid Mixture) in oligotrophic seawater was also tested. Cultures were grown in media amended with 1 m M S 2 O 3 2 , 100 μ M NO 3 − and an amino acid mixture (5 μ M of N) ( Supplementary Figure 4 ). There was no discernable difference between cultures amended with amino acids and unamended controls, indicating that strain EF1 prefers NH 4 + for biogenic nitrogen. Roles in the marine nitrogen cycle Evidence that cultured SUP05 produce NO 2 − suggests that related strains are a potential source of NO 2 − in OMZs. There is strong molecular evidence indicating that environmental SUP05 are capable of mediating sequential steps in denitrification. Because these environmental sequence data provide a fragmented view of a population of cells, it is also possible that different SUP05 cells carry out different steps in denitrification, depending on the diversity of SUP05 in a population, on the concentrations of substrates, and on the range of interactions within a community. Genetic and physiology data from this study suggest that a single strain of SUP05 carries out two non-sequential steps in denitrification. Both cultivation-dependent and cultivation-independent data indicate that the first step in denitrification (NO 3 − reduction to NO 2 − ) is highly conserved ( Canfield et al. , 2010 ; Walsh et al. , 2009 ; Hawley et al. , 2014 ; Murillo et al. , 2014 ), while the potential to carry out subsequent steps in denitrification are not always identified ( Murillo et al. , 2014 ). In addition, SUP05 are often most abundant in areas where NO 2 − accumulates ( Canfield et al. , 2010 ; Hawley et al. , 2014 ; Murillo et al. , 2014 ). Glaubitz et al. (2013) reported a positive correlation between SUP05 and nitrite concentrations in the Black Sea. Although heterotrophic NO 3 − respiration is currently considered the primary process leading to NO 2 − accumulation within OMZs, there is ample genetic and physiological data suggesting that sulfur-oxidizing chemoautotrophs from the SUP05 clade are a potential source of NO 2 − , fueling competing nitrogen removal processes in the ocean. “ Ca. T. autotrophicus” strain EF1 growth was also limited by NH 4 + . In the mid 1950s, Baalsrud and Baalsrud, and van Niel found that the S-oxidizing and NO 3 − -reducing bacterium Thiobacillus denitrificans required NH 4 + or amino acids for biosynthesis ( Baalsrud and Baalsrud, 1954 ; van Niel, 1955 ). Genes for NH 4 + transport and amino acid assimilation were identified in the strain EF1 genome and in a SUP05 metagenome assembled from Saanich Inlet ( Walsh et al. , 2009 ). They were also expressed by SUP05 in Saanich Inlet and the Southern Ocean ( Wilkins et al. , 2013 ; Hawley et al. , 2014 ). The biogenic nitrogen requirement of strain EF1 is low. For example, if “ Ca. T. autotrophicus” strain EF1 cells have 10 fg of carbon/cell and are at Redfield ratios for carbon and nitrogen (106:16), we estimate that they required ~0.22 μ M of nitrogen for biosynthesis in cultures grown on natural seawater media. By comparison, they reduced ~30 μ M of NO 3 − to ~30 μ M NO 2 − during respiration. There was no evidence that they respired NO 2 − or that they used amino acids instead of NH 4 + for biosynthesis ( Supplementary Figure 4B ). These data support the conclusion that strain EF1 requires relatively high concentrations of NO 3 − for respiration and relatively low concentrations of NH 4 + for biosynthesis. “ Ca. T. autotrophicus” strain EF1 also produced N 2 O. Members of the SUP05 bacteria expressed genes to produce N 2 O in regions of significant N 2 O cycling and emission ( Codispoti et al. , 2001 ). A recent study by Babbin and colleagues ( Babbin et al. , 2015 ) suggests that rapid N 2 O cycling in the suboxic ocean could lead to future increases in N 2 O emissions. Environmental sequence data indicate that SUP05 are broadly distributed and abundant in these regions. Growth experiments support field expression data, suggesting that N 2 O producing chemoautotrophic SUP05 have important roles in biologically driven nitrogen loss from the ocean ( Walsh et al. , 2009 ; Hawley et al. , 2014 ; Murillo et al. , 2014 )."
} | 5,319 |
32595437 | PMC7247826 | pmc | 6,305 | {
"abstract": "We demonstrate a variety of biologically relevant dynamical behaviors building on a recently introduced ultra-compact neuron (UCN) model. We provide the detailed circuits which all share a common basic block that realizes the leaky-integrate-and-fire (LIF) spiking behavior. All circuits have a small number of active components and the basic block has only three, two transistors and a silicon controlled rectifier (SCR). We also demonstrate that numerical simulations can faithfully represent the variety of spiking behavior and can be used for further exploration of dynamical behaviors. Taking Izhikevich’s set of biologically relevant behaviors as a reference, our work demonstrates that a circuit of a LIF neuron model can be used as a basis to implement a large variety of relevant spiking patterns. These behaviors may be useful to construct neural networks that can capture complex brain dynamics or may also be useful for artificial intelligence applications. Our UCN model can therefore be considered the electronic circuit counterpart of Izhikevich’s (2003) mathematical neuron model, sharing its two seemingly contradicting features, extreme simplicity and rich dynamical behavior.",
"introduction": "Introduction In his 2003 landmark paper Izhikevich (2003) emphasized that to develop a large-scale model of the brain one faces seemingly mutually exclusive requirements: on one hand the model had to be simple enough to allow for efficient computation and, on the other, it had to be able to produce a rich variety of biologically relevant firing patterns. Interestingly, the same dilemma is encountered for the implementation of neurons in neuromorphic circuits – i.e., circuits that perform computations based on the architecture of the brain. Recently we proposed an ultra-compact neuron (UCN) circuit that realizes the leaky integrate and fire model ( Rozenberg et al., 2019 ). That circuit complies with the first requirement, as it was simply based on only three active devices, two transistors and a thyristor, or SCR, plus a capacitor and a few resistors. Here we shall show that the UCN model also complies with the second requirement. Specifically, we shall demonstrate that the UCN is a circuit block that, with minimal variations, may realize at least 12 out of the 20 biological relevant behaviors highlighted by Izhikevich (2004) . To reproduce those behaviors has become a de facto standard to demonstrate the relevance of a spiking neuron model implemented on different physical supports. The literature is very diverse and growing fast, so we shall only cite a few examples here and refer the readers to further references in those works and in the review of Indiveri et al. (2011) : the digital processor chips TrueNorth developed by IBM ( Cassidy et al., 2013 ; Merolla et al., 2014 ) and the more recent ODIN by ICTEAM ( Frenkel et al., 2019 ); the compact neuron circuit, with only 14 MOSFET transistors proposed by Wijekoon and Dudek (2008) ; or the radically different spiking neuron based on vanadium dioxide ( Yi et al., 2018 ), a Mott insulator memristive material ( del Valle et al., 2018 , del Valle et al., 2019 ). Other interesting proposals, which aimed at a faithful physical implementation of the Izhikevich mathematical model equations are: a compact circuit of MOS transistors in the subthreshold regime, simulated with MOSIS libraries ( Rangan et al., 2010 ); a CMOS digital neuron for event-driven computation, simulated in Spice ( Imam et al., 2010 ). Silicon Neuron (SiN) Circuits The UCN belongs to the class of SiN circuits ( Indiveri et al., 2011 ), which are electronic hardware implementations of systems that aim to emulate the electric behavior of biological neurons. These SiN blocks may then be integrated to construct larger circuits ( Qiao et al., 2015 ), such as to emulate neural network for artificial intelligence applications, or brain-like systems for basic neuroscience research. The SiN circuits are inspired from a multiplicity of mathematical neuronal models that range from the simplest integrate and fire to the realistic Hodgkin-Huxley ( Gerstner et al., 2014 ). Depending on the desired goal, SiN implementations may favor different features, such as low power dissipation, circuit simplicity, low variability, realistic behavior, tunability, etc. Typically, they are implemented using CMOS technology and VLSI ( Indiveri et al., 2011 ), and they can be broadly classified as sub-threshold or above-threshold depending on the conduction mode of the transistors. The sub-threshold systems follow from the pioneer work of Mead ( Mead, 1989 , Mead, 1990 ) and of Mahowald and Douglas (1991) that emphasized the similarity between the exponential behavior of carrier conduction in transistor channels with that of ionic channels in neurons, and coined the concept of “neuromorphic behavior.” The systems in the sub-threshold regime have the additional attractive features of low power dissipation, which follows from the small currents, and time constants that are compatible with the biological ones ( Indiveri et al., 2011 ). However, a main drawback is the so called device mismatch, which is a relatively large variability between cells ( Indiveri et al., 2011 ). As an example of this approach we may mention that of Yu and Cauwenberghs (2010) that implemented a SiN to realize the realistic Hodgkin-Huxley model. The above-thershold implementations avoid mismatch and thus have the precision needed to faithfully recreate the mathematical models that motivate them. These SiN circuits also operate a time-constants that are much faster (10 3 –10 4 ) than the biological ones, unless they adopt off-chip larger capacitors. One example of above-threshold systems is the implementation of a tunable Hodgkin-Huxley model by Saighi et al. (2011) . A different approach is to develop SiN circuits that are motivated on generalizations of the simple integrate and fire model ( Gerstner et al., 2014 ). Some examples are the AdEx ( Brette and Gerstner, 2005 ), the Izhikevich ( Izhikevich, 2003 , 2004 ) and the Mihalas-Niebur neuron ( Mihalas and Niebur, 2009 ). These models do not necessarily have a biological underpinning as the Hodgkin-Huxley, but nevertheless were shown to capture the relevant spiking patterns observed in biological neurons. Their main attractive is that their relative simplicity allow for more efficient implementations in both software and hardware ( Wijekoon and Dudek, 2008 ; Folowosele et al., 2009 ; Livi and Indiveri, 2009 ; Indiveri et al., 2010 ; Rangan et al., 2010 ; van Schaik et al., 2010a , b ; Qiao et al., 2015 ). The Ultra-Compact Neuron In this context the UCN that we introduced recently ( Rozenberg et al., 2019 ) opens a different paradigm. Similar to sub-threshold systems and faithful to the concept of neuromorphic engineering, it exploits an intrinsic non-linearity of an electronic device. Namely the threshold switching of the SCR conductance emulates the firing of biological neurons. As we discussed in Rozenberg et al. (2019) , this features permits a drastic reduction to the number of components to implement a basic leaky-integrate-and-fire (LIF) SiN. So in this regard it may be considered as belonging to the class of Compact SiN circuits ( Indiveri et al., 2011 ). However an attractive feature of the UCN is that they can be directly interconnected, therefore need not a priori require an additional address-event representation off-chip system. Despite the fact that the thyristor was introduced in the very beginnings of semiconductor electronics, it is currently not a conventional CMOS device. Its development in microelectronics is mostly restricted to protection circuits ( Ker and Hsu, 2005 ), which nevertheless demonstrates that there are no a priori impediments for its CMOS implementation. The time-constants associated to the switching of a SCR are short, thus in this regard the operation of the UCN follows similar features as the above-threshold SiN as we mentioned above. Therefore, if the goal is to achieve biological time-scales one may need large “membrane” capacitors, hence our UCN should not be considered compact in regard of the wafer real estate. In the present work we shall describe how the functionality of the basic UCN block can be extended to realize a variety of biologically relevant spiking patterns, without a sacrifice of circuit simplicity. The paper is organized as follows: In section Materials and Methods we shall describe our recently introduced UCN circuit ( Rozenberg et al., 2019 ). We shall demonstrate how the basic behavior of the UCN can be very precisely captured by means of numerical simulations obtained with LTspice ( LTspice ® , 2020 that we validate against actual circuit measured data. Section Results contain the main results of the present work. In the first part, we exploit the simplicity of the simulation package capabilities to explore extensions of the basic UCN circuit block, searching for different types of biologically relevant dynamical behaviors. We shall demonstrate that small variants of a basic circuit allow us to capture at least 12 out of the 20 dynamical behaviors, including some inhibition ones. In the second part of this section, we use the simulation results to achieve the main goal of this work, namely to provide the explicit circuits and measure them to demonstrate that the thyristor-based UCN can realize the complex firing patterns observed in biological systems. Our simulations inform and guide the implementation of the actual electronic circuits that we construct with out-of-the-shelf components. This feature underscores the relative ease for the reproducibility of our work and may prompt other research groups to embark along the present line of work. The circuits details and the list of components are described in the Supplementary Material . In section Discussion we finally discuss some specific technical aspects of our work in regard of different open challenges in the field.",
"discussion": "Discussion In this work we have illustrated the versatility of the UCN circuit to capture a significant number of biologically relevant neuronal behaviors. We have not attempted to demonstrate the totality of the 20 behaviors identified by Izhikevich (2004) . Rather, our goal was to demonstrate that the minimal UCN circuit is a sound basis to implement a new type of spiking neuron model of remarkable simplicity. Another important result of our study was the successful implementation of the numerical simulation package to efficiently search for the complex spiking patterns. This required the implementation of the SCR model. More generally, reliable numerical simulations methods become an essential tool to implement small neural sub-circuits counting tens or hundreds of spiking neurons. The key feature that enables this circuit simplicity is the memristive behavior of the SCR, which is a conventional electronic component that may be implemented in CMOS technology ( Ker and Hsu, 2005 ; Tong et al., 2012 ). However, we should also note that although the UCN model and the extensions proposed in the present work only require a reduced number of electronic components, they are of different types, which may pose a challenge for the integration into a single technology. Nevertheless, this may be achieved by Bi-CMOS ( Alvarez, 1990 ), or the more recent BCD8sP technology ( Roggero et al., 2013 ). Simulation of our UCN circuits to implement actual chips is an exciting prospect that is beyond the scope of the present work. Also in regard of the prospects for microelectronic implementation, one should be aware that, similarly to all compact neuron model circuits based on standard electronics, the UCN also requires a “membrane” capacitor to integrate charge. This feature remains a significant problem for miniaturization as the capacitors still require a relatively large physical space in VLSI. The ultimate solution for a low-power and low-footprint spiking neuron device may therefore require memristors based on Mott materials ( del Valle et al., 2018 ). However, achieving a reliable control and a theoretical understanding of the metal-insulator transitions in those compounds still represent a significant challenge ( Yi et al., 2018 ; del Valle et al., 2019 ). To conclude, the UCN model is a simple modular block that can be used to implement spiking neuron circuits. The present work demonstrates that its simplicity does not prevent the realization of complex spiking patterns, beyond the integrate and fire paradigm. Our work opens a new way for the implementation of large neuronal networks with biological plausibility and of unprecedented simplicity."
} | 3,179 |
25188470 | PMC4154735 | pmc | 6,306 | {
"abstract": "To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus , followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg −1 ) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg −1 cadmium whereas only one strain tolerated 1,000 mg kg −1 cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml −1 . In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml −1 ) with two Bacillus sp. producing more than 100 µg ml −1 . Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.",
"introduction": "Introduction Mining industry has caused extensive environmental and public health problems [1] – [3] . A wide variety of heavy metals such as zinc, lead, copper and cadmium have been detected in soil at mining sites presenting a major threat to the environment and population [4] , [5] . Heavy metals cannot be biologically degraded and indefinitely persist in the environment. Heavy metals transferred through the food chain are a serious hazard to human health [6] . Due to contamination by heavy metals, mining sites are surrounded by large barren areas. The awareness of the detrimental heavy metal contamination at mining sites has increased in recent years. The toxic heavy metals accumulated in soil can effectively impact the microbial community composition. Bacteria play an important role in maintaining soil fertility and structure. Because bacteria respond quickly and are sensitive to subtle environmental changes, they have been considered as efficient bio-indicators of soil quality [7] . Both the structural and functional bacterial diversity are important indicators of soil health [8] . Phytoremediation has been effectively used to remediate heavy metal-polluted sites as a sustainable remediation approach [9] . Plant-microbe partnerships may be utilized to improve biomass production and remediation [10] . Plant growth-promoting rhizobacteria (PGPR) that solubilize phosphate and synthesize growth-promoting substances such as indoleacetic acid (IAA) and siderophores can be applied in the plant-assisted bioremediation of metal-contaminated soil [11] – [14] . Phytoremediation utilizes heavy metal-tolerant plant species with metal accumulation ability. Since the addition of IAA to soil can enhance the uptake of metals in plant roots [15] , [16] , bacteria-producing IAA have been used to assist the phytoremediation of soil contaminated with heavy metals [17] . Metals such as iron, zinc, copper, manganese and nickel play important roles as essential or beneficial micronutrients of microorganisms [18] , [19] . However, a high concentration of metal ions in soil shows serious effects on microbial communities by changing the community structure and decreasing diversity and total microbial biomass [13] . Therefore, microbial communities are useful indicators of the effect of contamination on soil health [20] . To have a functional role in remediation, bacteria in heavy metal-contaminated soil must first overcome the heavy metal stress. Microorganisms tolerate heavy metals by immobilizing metals on cell surfaces or transforming metals into less toxic forms, for example by precipitation, acidification and oxidation-reduction [21] . Panzhihua is an industrial and mining city in Sichuan of Southwest China with over 10 9 tonnes of ore reserves deposited as iron-vanadium-titanium oxide (V-Ti magnetite) [22] . The world class magmatic deposits of V-Ti magnetite in Panzhihua provide 20% of iron (Fe), 64% of vanadium (V) and 53% of titanium (Ti) supply for China [23] . Long-term mining activities have contaminated soils and sediments in Panzhihua by metals, especially by V, Ti and Fe [24] . The redox-sensitive vanadium is toxic to soil microorganisms and plants [25] . Even though titanium is beneficial to plants at low concentrations, high concentrations of titanium are toxic [26] . Iron is an essential nutrient serving as a catalyst for many cellular reactions, in particular those involving redox and O 2 chemistry [27] . More than 220 million m 3 of mine tailing has been piled up in Zhujiabaobao, Panzhihua, creating a serious environmental hazard. Because the mine tailing soil contains heavy metals, only few plants grow on it, creating large barren areas. Long term exposure to contaminant allows different bacteria to become adapted to the contaminant, making autochthonous bacteria more useful in bioremediating the contaminated environment compared to allochthonous ones [28] . Therefore, this study focuses on the culturable heavy metal-resistant and plant growth-promoting autochthonous bacteria from V-Ti magnetite mine tailing soil, with the aim of providing information for bioremediating the large area covered by the V-Ti magnetite mine tailing dam at Panzhihua.",
"discussion": "Discussion Basic physicochemical properties of the V-Ti magnetite mine tailing soil The basic physicochemical properties are important factors for evaluating soil quality. The V-Ti magnetite tailing, a weakly acid soil, showed pH similar to that in an old Spanish Pb-Zn mine soil [42] . Soil pH is the best predictor of microbial diversity and community composition [43] , [44] . The bacteria in the V-Ti magnetite mine tailing soil may prefer acid environment. The organic matter content of V-Ti magnetite mine tailing soil was lower than in agricultural and urban ecosystem soils [29] , [45] , but similar with other mine tailing area [42] . The V-Ti magnetite mine tailing also showed lower content of available N, P and K than agricultural and urban ecosystem soils [29] , [45] , The low contents of available N, P, K and organic matter suggested that V-Ti magnetite mine tailing soil was unfertile. The iron, titanium and vanadium concentrations were up to three, ten and 70 times higher than in US soils in average [46] , respectively. The concentrations of Cr, Zn, Cu, Ni and Mn were approximately 1.5 to 2.5 times higher than in US soils in average [46] . The concentrations of Fe, Cu and Zn were above values considered very high by Abreu et al \n [47] and the chromium concentration was almost twice as high as needed to inhibit alfalfa germination [48] , plausibly explaining the scarce vegetation at the Zhujiabaobao V-Ti magnetite tailing dam. Therefore, phytoremediation of the barren V-Ti magnetite mine tailing soil should include increasing the content of available N, P, K and organic matter and lowing metal concentration. Genetic identification of bacterial isolates To assess if the heavy metal-resistance and plant growth promoter-producing bacteria in the Zhujiabaobao V-Ti magnetite tailing dam soil would support phytoremediation, we isolated 136 bacterial strains, grouped them by BOX A1R-PCR and identified representative strains by 16S rRNA gene sequencing. The bacteria in the V-Ti magnetite mine tailing soil represented both Gram-negative and Gram-positive species. Bacillus spp. were the most abundant species, followed by Rhizobium spp. and Ochrobactrum spp. The spore and cyst forming capability of Bacillus spp. may explain why Bacillus spp. were abundant in the unfavorable environment of the V-Ti magnetite mine tailing soil. Autochthonous Bacillus from mine tailing in South Korea showed the ability to biomineralize heavy metals, such as Pb and Cr [49] , [50] . Ochrobactrum spp. and Pseudomonas spp. have been used for the bioremediation of environmental pollutants [51] – [53] . These observations suggested that the indigenous bacteria might be useful for phytoremediation of the Zhujiabaobao mine tailing soil. The presence of multiple autochthonous Rhizobium spp. implied that, with compatible leguminous plants, the rhizobium-legume symbiosis could be used to gradually increase nitrogen content and overall fertility in the barren soil. The symbiosis of rhizobia and leguminous plants has been effectively used to remediate contaminated soil [54] , [55] . Heavy metal tolerance of bacterial isolates To estimate the usefulness of the isolated bacteria in bioremediation, we assessed their heavy metal tolerance. Obviously, the bacteria in V-Ti magnetite mine soil have to tolerate the harsh environment polluted by heavy metals. The tolerance mechanisms include exclusion, extrusion, accommodation, bio-transformation and methylation or demethylation [13] . Bacteria can enhance metal solubility by producing acid and detoxify metals by removal, sequestering or immobilizing [56] . Since heavy metal tolerance is one of the most important factors for using an indigenous microorganism in bioremediation, recently the functional diversity of bacterial communities in contaminated soil has attracted more attention [7] . The bacterial isolates from V-Ti magnetite mine tailing soil showed diverse tolerance to different heavy metals. Most of the isolates did not tolerate the lowest tested concentration (200 mg kg −1 ) of Ni, Co and Zn. The concentrations of Ni and Zn were low in the mine tailing soil. A few of the V-Ti magnetite mine tailing isolates, e.g. Bacillus sp. KT87 and Bacillus sp. KT72, showed tolerance to higher metal concentrations than isolates from a copper mine tailing, from a mercuric salt-contaminated soil and from chickpea rhizosphere soil [57] – [59] . Interestingly, even though the concentration of Cd in the mine soil was not high, more than half the isolates tolerated a high concentration of Cd (1,000 mg kg −1 ). Likewise, even though the concentration of Pb in the mine tailing soil was low, some isolates tolerated a high concentration of Pb, i.e. the tolerance to heavy metals and the heavy metal content of soil did not directly correlate. Many mine tailing sites are polluted by multiple metals. For bacteria, the ability to survive, including variation in strains and characteristics, is related to environmental conditions and length of exposure to those conditions [60] . Therefore, the isolates from the V-Ti magnetite mine tailing soil showing multi-metal resistance were affected by the unfavorable environment. The culturable bacteria included isolates with multiple heavy metal tolerance, especially among the Bacillus spp., suggesting that the indigenous bacteria are capable of assisting the bioremediation of the V-Ti magnetite mine tailing soil polluted by heavy metals. Plant growth-promoting activity of bacteria Plant-associated bacteria play a key role in host adaptation to changing environment by altering plant cell metabolism or promoting plant growth. Plant growth promoting rhizobacteria (PGPR) producing IAA and siderophore have been widely used to accelerate phytoremediation of metalliferous soil [13] , [21] . Production of indoleacetic acid (IAA), a phytohormone, is a key characteristic of PGPR [61] . The addition of IAA to soil can enhance the uptake of metals in plant roots [13] , [15] , [62] . Even though PGPR are widely studied, few studies have systematically analyzed PGPR in contaminated soil. About 23% and 50% of Zn- and Cd-accumulating isolates from a former zinc and lead mining and processing site in Austria produced IAA and siderophore, respectively [63] . In V-Ti magnetite mine tailing soil the percentages of culturable rhizosphere IAA and siderophore producers were entirely different at 67% and 21%, respectively. The plant growth promoting activity of the isolates from the V-Ti magnetite mine tailing was stronger than that reported for Pseudomonas putida GR12-2 (IAA: 2.01 µg ml −1 ) from the rhizosphere of an arctic plant [64] , but lower than that of Alcaligenes faecalis BCCM IC 2374 (Siderophore: 347 µg ml −1 ) [65] , suggesting that the plant growth promoting activity from different environments is totally different. The abundance of isolates producing more than 20 µg ml −1 IAA suggested that the plant growth promoting ability of the isolates might assist in phytoremediating the soil. The bacterial and fungal siderophores facilitate iron uptake in soil [10] . Iron chelated by siderophores is unavailable to plant pathogens resulting in an increase in plant health [13] . Metal–resistant siderophore-producing bacteria play important role in the successful survival and growth of plants in contaminated soil by alleviating metal toxicity and supplying nutrients for plant, and bacterial siderophore can bind metals other than iron [66] , which may be the reason why microorganism can survive in the mine tailing soil contaminated by multi-metals. Bacterial siderophore should be beneficial to regulate availability of the abundant iron in the V-Ti magnetite mine tailing soil containing high concentration of iron. As bioaugmentation-assisted phytoextraction technology, the indigenous siderophore-producing bacteria can increase the phytoextraction rate that usually limits the use of phytoremediation methods [67] . Aside from their involvement in iron acquisition, siderophores have physiological roles of protecting some bacteria against the toxic effect of pyochelin by reducing reactive oxygen species [68] , so the presence of siderophore-producing bacteria in the mine tailing can directly or indirectly promote bioremediation for the contaminated soil. Many isolates showed both IAA and siderophore production, implying that the characteristics of the indigenous bacteria are helpful in bioremediating the desert mine tailing area. Phytoremediation of metals was facilitated by PGPR by promoting plant growth and increasing the amount of metal taken up by plant [21] , [69] . The heavy metal-resistance and plant growth-promoting activity are key characteristics for bacteria that are to be applied in metal phytoremediation. Therefore, analyzing these characteristics in an indigenous bacterial in contaminated sites is essential to provide significant information for developing effective bioremediation measures. Moreover, because both the structural and functional bacterial diversity are important indicators of soil health, evaluation for diversity of heavy metal-resistant bacteria and PGPR should be considered as the primary work for bioremediating soil contaminated by heavy metals. We showed that V-Ti magnetite mine tailing soil in Zhujiabaobao contained abundant bacteria that tolerate multiple heavy metals and have plant growth-promoting abilities. The results suggested that the indigenous bacteria in the soil have characteristics beneficial for remediating the contaminated mine tailing soil. To further study the phytoremediation approach, the plant growth promoting activity will be studied both in greenhouses and in situ in Zhujiabaobao."
} | 3,833 |
38030903 | PMC10730404 | pmc | 6,307 | {
"abstract": "Alternative solutions to mineral fertilizers and pesticides that reduce the environmental impact of agriculture are urgently needed. Arbuscular mycorrhizal fungi (AMF) can enhance plant nutrient uptake and reduce plant stress; yet, large-scale field inoculation trials with AMF are missing, and so far, results remain unpredictable. We conducted on-farm experiments in 54 fields in Switzerland and quantified the effects on maize growth. Growth response to AMF inoculation was highly variable, ranging from −12% to +40%. With few soil parameters and mainly soil microbiome indicators, we could successfully predict 86% of the variation in plant growth response to inoculation. The abundance of pathogenic fungi, rather than nutrient availability, best predicted (33%) AMF inoculation success. Our results indicate that soil microbiome indicators offer a sustainable biotechnological perspective to predict inoculation success at the beginning of the growing season. This predictability increases the profitability of microbiome engineering as a tool for sustainable agricultural management.",
"discussion": "Discussion Here we show that inoculation with arbuscular mycorrhizal fungi significantly increased maize yield. We achieved a significant positive increase in biomass of 12–40% in a quarter of the fields, which is considerably higher than the annual yield increases through breeding for a range of crops (which are often below 1%) 32 . Moreover, effect sizes of adding cover crops (up to 8%) 33 and other biofertilizers (up to 12%) 34 in comparable climatic regions and production systems are also lower compared with growth increases in inoculated high-MGR fields in this study. While many studies pointed to the importance of AMF for plant nutrition, this study links AMF inoculation to soil pathogen protection. Pathogen abundance in the soil best explained AMF inoculation success (33% of variance explained), while soil parameters were less important (29%; Fig. 5a ). While a range of studies have shown that inoculation with AMF can promote plant growth in the field 9 – 11 , results are variable and none have used soil characteristics and molecular-based soil microbiome analysis to specifically predict under which conditions AMF can promote plant growth. Phosphorus availability tended to be negatively associated with inoculation success in previous studies 35 . In our study, phosphorus explained less than 2% of the variation in MGR, which was also reflected in the outcome of the fertilizer trial (see Supplementary Results and Extended Data Fig. 1 ). Despite a large (factor of 26) variation in immediately plant-available phosphorus levels (0.34–9.07 mg kg −1 , H 2 O-CO 2 extraction; Supplementary Table 1 ), most soils were above the threshold for phosphorus deficiency in Swiss soils (0.58 mg kg −1 ) 36 , perhaps also explaining why AMF inoculation success was best explained by other factors. Further, positive growth responses were associated with lower soil organic carbon levels and especially with reduced soil microbial biomass carbon (Fig. 5b ). Soil microbial biomass represents the living fraction of organic carbon and is an important component of soil health 37 , 38 . Fields with low microbial carbon content appeared to benefit more from AMF inoculations, suggesting that AMF are particularly important when soil health is low. It is also known that organic amendments can suppress a wide range of pathogens in the soil 39 , 40 . Therefore, protection from pathogens by inoculated AMF may be particularly important in soils with low organic content. Consequently, AMF inoculations in healthier soils with high abundance of OTUs associated with low MGR (for example, Phaeohelotium ; Supplementary Table 10 ) are less likely to provide economic benefits. Several sOTUs associated with high MGR in this study are known as plant pathogenic taxa (Supplementary Table 10 ) and can infect important crops including maize 41 – 45 . These comprise Olpidium brassicae 41 , 42 , Myrothecium sp. 43 and Fusarium equiseti 44 , 45 . The most important predictor in the model, however, was sOTU18 with the genus assignment Trichosporon , known to cause diseases in human 46 . So far, this genus has not been described in relation to plant pathogenicity; yet, it best explained inoculation success with AMF and especially in high-MGR fields where it was less abundant in inoculated plots, AMF had a positive impact on plant yield (Supplementary Tables 14 and 15 ), suggesting a negative effect of this taxon on maize growth. Moreover, sOTU18 is an indicator of poorer soil properties (that is, negatively correlated with organic carbon and soil fertility, and positively correlated with sand content; Extended Data Fig. 8 ). Overall, pathogen abundance might be more pronounced in poorer soil. The addition of AMF provides additional protection and plants growing in these fields might benefit more from mycorrhizal inoculation. Given the limitations of marker genes in predicting fungal lifestyles, further studies need to isolate these fungi and test whether they indeed negatively affect maize growth to experimentally verify their pathogenicity potential and to what extent AMF can contribute to pathobiome management. Only few pathogens seem to be important in the studied context, as the summed abundances of all soil fungal pathogens identified by guild-based screening was not able to predict MGR (see Supplementary Results and Extended Data Fig. 4 ). AMF strains are probably specialized in their ability to protect against specific pathogens. Here we inoculated an AMF strain that was isolated from Swiss soil and can establish well in a wide range of soil types 24 , 35 . The inclusion of other AMF genotypes to be screened for their properties to protect against specific pathogens would not only broaden the scope of this management practice and facilitate establishment under a wide range of conditions, but could also prevent possible agricultural intensification and biodiversity loss through the employment of only one AMF strain. Even though we did not observe a reduction in AMF diversity (Supplementary Table 16 ), future studies need to investigate the long-term effects of inoculations, as well as the persistence and invasiveness of native vs exotic AMF inocula. The unintended consequences of non-native inoculants in natural and agricultural systems are not known, but if inoculants are invasive, they may pose a threat to soil and plant biodiversity and ecosystem functioning 47 . Furthermore, complementary to more diverse and complex inocula, the possibility of AMF rotations—analogous to crop rotations—could also mitigate the risk of low-diversity microbial treatments on soil biodiversity. The ability of AMF to protect plant roots from attack by soil-borne pathogens can be explained by various mechanisms including improved plant nutrient uptake and consequently plant health 48 , induced systemic resistance 49 , alteration of the root microbiome 50 and direct competition for root space 18 , 19 , 51 . In our study, several of these mechanisms of action probably occurred simultaneously. Our root microbiome data partly point to direct competition for root colonization. In fields with high MGR, pathogenic fungi were significantly less abundant in inoculated roots (Fig. 6 ). These included the previously identified important soil pathogens Olpidium and (potentially plant pathogenic) Trichosporon , as well as Cladosporium , Mycochaetophora , Pyrenochaeta and Vishniacozyma . Myrothecium and Fusarium , which were also identified as important predictors, could not be found in the root microbiome data, possibly because the molecular primers used for roots were specifically designed to target AMF 52 . Thus, general ITS (internal transcribed spacer) primers for the roots also need to be included in future studies to cover full fungal diversity. Moreover, it was striking that there was no correlation between root colonization and plant growth response. Inoculation of the AMF strain SAF22 was the experimental factor, but inoculum success and how well the AMF strain established was not a good predictor. Instead, differences in its functions explained the variation in MGR. In contrast to the common interpretation where biofertilizers stimulate plant growth, here the interpretation is the other way around: abundant pathogenic soil fungi, which are present in ‘high-MGR’ fields, cause a growth reduction in the control treatment, while this otherwise negative effect is mitigated by the inoculated AMF. We believe this could be due to several reasons closely related to the many ways AMF can suppress pathogens. First, if AMF establish first and fast, this could prevent or reduce pathogen establishment. Second, a range of studies have shown that AMF can trigger induced systemic resistance 49 , 53 – 55 , and AMF may indirectly affect pathogens by altering the microbiome 50 . Time-resolved studies that follow the processes and mechanisms in the roots throughout the growing season are needed. Several studies have shown that the abundance and activity of AMF are also explained by the bacterial microbiome 56 and pesticide application 57 , 58 . Further, differences in microclimatic conditions may be another factor contributing to differences in MGR. The inclusion of such factors may resolve even more of the unexplained variance. However, while field inoculations must be economically viable, simple and cost-effective prediction of inoculation success must also be possible. Predicting MGR based solely on sequencing soil fungal pathogens, for instance, would represent a simplified diagnostic approach. Here we present an initial list of pathogenic sOTUs that could be quantified directly in the field at the beginning of the growth season, with results being available within a few hours and at a reasonable cost using quantitative PCR or rapid sequencing 59 . Furthermore, automated and affordable microbial diagnostic assays could be developed (for example, Loop-mediated isothermal amplification). Subsequently, pathogen abundance can predict inoculation success. With this work using 54 fields, we have shown that field inoculation with AMF can successfully be predicted and can give a yes/no recommendation with high accuracy of 80–83%; this means a successful prediction in 4 out of 5 fields. We have solved the context dependency for one maize variety in one geographic area. The approach presented here is easily transferable and further studies need to test different maize varieties, as their responsiveness to mycorrhiza can vary greatly 23 , 60 . The inclusion of a broad range of soil types and climatic zones will further extend the scope of the work. To maximize the potential of AMF for more sustainable agricultural production systems, future work needs to include settings with reduced use of agrochemicals. Furthermore, our approach can be used as a blueprint to predict inoculation success and resolve context dependency of other widely used biofertilizers including Rhizobium spp. or Bacillus amyloliquefaciens 61 . With our results, we provide a crucial starting point for the development of a diagnostic tool using soil microbial indicators that can ultimately increase the reliability of field inoculations. As a result, AMF inoculations can become a powerful management option for microbiome engineering in arable land and thus an integral part of agricultural sustainability."
} | 2,876 |
33889135 | PMC8057349 | pmc | 6,308 | {
"abstract": "Rhizosphere microbial communities are known to be related to plant health; using such an association for crop management requires a better understanding of this relationship. We investigated rhizosphere microbiomes associated with Verticillium wilt symptoms in two cotton cultivars. Microbial communities were profiled by amplicon sequencing, with the total bacterial and fungal DNA quantified by quantitative polymerase chain reaction based on the respective 16S and internal transcribed spacer primers. Although the level of V. dahliae inoculum was higher in the rhizosphere of diseased plants than in the healthy plants, such a difference explained only a small proportion of variation in wilt severities. Compared to healthy plants, the diseased plants had much higher total fungal/bacterial biomass ratio, as represented by quantified total fungal or bacterial DNA. The variability in the fungal/bacterial biomass ratio was much smaller than variability in either fungal or bacterial total biomass among samples within diseased or healthy plants. Diseased plants generally had lower bacterial alpha diversity in their rhizosphere, but such differences in the fungal alpha diversity depended on cultivars. There were large differences in both fungal and bacterial communities between diseased and healthy plants. Many rhizosphere microbial groups differed in their abundance between healthy and diseased plants. There was a decrease in arbuscular mycorrhizal fungi and an increase in several plant pathogen and saprophyte guilds in diseased plants. These findings suggested that V . dahliae infection of roots led to considerable changes in rhizosphere microbial communities, with large increases in saprophytic fungi and reduction in bacterial community.",
"conclusion": "Conclusion This study demonstrated that V . dahliae infection and subsequent disease development can lead to large changes in rhizosphere microbiomes in cotton. In addition to high V . dahliae inoculum density, a high fungal/bacterial biomass ratio in rhizosphere is an indicator of wilt disease development. Much of the increased fungal abundance in diseased roots is likely contributable to increased fungal saprophytes.",
"introduction": "Introduction Cotton ( Gossypium hirsutum L.) is an important fiber crop. Cotton Verticillium wilt, caused by Verticillium dahliae , is one of the most devastating plant diseases worldwide (Klosterman et al., 2009 ). The pathogen can survive in the soil as resting microsclerotia without a host plant for more than 14 years. In China, its hosts include a number of economically important crops such as potato ( Solanum tuberosum L.), tomato ( Lycopersicon esculentum Miller), strawberry ( Fragaria × ananassa ), sunflower ( Helianthus annuus ), eggplant ( Solanum melongena L.), and pepper ( Capsicum annuum L.). Incidence of cotton wilt increases with increasing densities of V. dahliae microsclerotia in soil (Wei et al., 2015 ). Controlling Verticillium wilt is difficult because of the inaccessibility of the pathogen during infection, long-term survival of microsclerotia in soil, and its broad host range (Klosterman et al., 2009 ). There has been limited success in planting resistant cultivars of upland cotton against wilt in heavy infested fields (Zhang et al., 2012b ). In Xinjiang, the main cotton production region in China, crop rotation with non-hosts of V . dahliae has not been adopted because of the difficulties in changing cropping systems and saline–alkali soils. Soil fumigation with methyl bromide was very effective against V. dahliae but has already been banned under the Montreal Protocol (Martin, 2003 ). Although several remaining fumigants, such as chloropicrin and dazomet, can be used to manage wilt, farmers in China are reluctant to use them in cotton production because of their limited economic benefits. Plants harbor diverse microbiota both inside and outside their tissues (Vandenkoornhuyse et al., 2015 ). Rhizosphere microbiota, which closely interact with plant roots, are important for plant health (Berendsen et al., 2012 ) and crop yield potential (Xu et al., 2015 ) and influenced by many factors such as plant species and developmental stage, soil properties, nutrient status, land use, and climatic conditions. Selective recruitment of specific microbes by plant roots has been observed (Peiffer et al., 2013 ; Bai et al., 2015 ; Zarraonaindia et al., 2015 ). Suppression of soilborne disease has long been considered as one of the key benefits associated with beneficial microbes in soil (Mendes et al., 2011 ; Bai et al., 2015 ; Finkel et al., 2017 ; Xiong et al., 2017 ). Microbial diversity and composition are related to plant disease resistance (Wei et al., 2019 ); high microbial diversity provides greater protection against soilborne pathogens (van Elsas et al., 2002 ; Mallon et al., 2015 ). Understanding the association of plant health with rhizosphere microbiota may provide a basis for manipulating soil microbiomes directly (e.g., amending soil with specific microbes) and/or indirectly (e.g., altering management practice) to promote plant health. Biocontrol of soilborne pathogens has long been a goal of sustainable agriculture, but because of the complexity of the soil environment and resident microbial communities, there are limited numbers of commercial biocontrol products against soilborne diseases in commercial agriculture (Mazzola and Freilich, 2017 ). To ensure that introduced microorganisms remain effective against pathogens over time in the soil environment, a clear understanding of how the introduced microbes interact with soilborne pathogens and other soil microorganisms is necessary. This knowledge may assist in development of cultural measures to increase the suppressiveness of soil microbiomes against soilborne pathogens and to improve survival (and hence enhance efficacy) of introduced biocontrol microbes. Multinutrient interactions among resident microbes may be disturbed by plant pathogens, which could cause community reorganization and lead to large-scale collapse and serious degradation of soil ecosystems (van der Putten et al., 2007 ). There is, however, limited knowledge on the changes in rhizosphere microbiota due to infection of plant roots by pathogens. Recently, several studies have shown that soilborne pathogens can significantly affect soil bacterial composition under field conditions (Shanmugam et al., 2011 ; Zhang et al., 2011 ; Wu et al., 2015 ) or in greenhouse (Mendes et al., 2011 ; Li et al., 2014 ). Specific fungal groups, e.g., Mortierella spp., may play an important role in the development of soil suppressiveness against Fusarium wilt disease in vanilla ( Vanilla planifolia ) (Xiong et al., 2017 ). However, bacterial and fungal communities are rarely investigated together to understand the nature of disease suppressive soil. The present study focuses on the changes in rhizosphere microbiome associated with the occurrence of Verticillium wilt on two cotton cultivars. Specifically, we quantified V . dahliae inoculum in rhizosphere soil and assessed the wilt severity for a number of pairs of plants (healthy and diseased plants) in two cultivars. Then we used amplicon metabarcoding to profile rhizosphere microbiome of these paired healthy–wilted plants and quantified the total biomass of fungi and bacteria DNA using quantitative polymerase chain reaction (qPCR) with generic internal transcribed spacer (ITS) and 16S primers. Finally, we established the differences in microbial communities in the rhizosphere between healthy and diseased plants.",
"discussion": "Discussion In the present study, similar to our previous finding (Wei et al., 2015 ), wilt severity increased significantly with the increasing V . dahliae inoculum density in the rhizosphere. However, the inoculum level explained only a very small proportion of the variability in observed wilt severities among plants, indicating that most differences in wilt severities are likely due to other factors rather than the differing inoculum densities. Total bacterial biomass, as indicted by qPCR results, was significantly higher in the rhizosphere of healthy plants than that of diseased plants; the opposite was true for the total fungal biomass. Similar results in total bacteria were also observed for potato plants with high and low levels of potato common scab severity (Shi et al., 2019 ). Suppressive soil against Fusarium wilt has higher populations of bacteria than the wilt-conducive soil that has higher populations of fungi (Peng et al., 1999 ). The occurrence of cotton Verticillium wilt appears to be accompanied by increased fungal abundance in the rhizosphere. The ratio of total fungi with total bacteria in the rhizosphere of wilt cotton plants is much > 1; the opposite is true for the healthy plants. Therefore, the ratio of total fungal to total bacterial biomass can be considered as an indicator of cotton Verticillium wilt occurrence. This agrees with previous findings that the ratio of fungi to bacteria shows increasing trends in the soil of continuous cropping that leads to severe soilborne disease of Panax notoginseng (Dong et al., 2016 ). Interestingly, this ratio appears to be less variable for healthy plants than for diseased plants, suggesting that healthy plants are associated with stable fungal/bacterial communities. Microorganisms are one indicator of soil health, particularly disease suppressiveness (Epelde et al., 2014 ; Ferris and Tuomisto, 2015 ; van Bruggen et al., 2015 ). High microbial diversity can improve community stability (Lefcheck et al., 2015 ; Delgado-Baquerizo et al., 2016 ). However, the relationship between microbial diversity and disease development varies with studies. For instance, a higher diversity in soil bacteria is associated with healthy plants in P . notoginseng (Wu et al., 2015 ) and cotton (Zhang et al., 2011 ), whereas the opposite was observed in tomato plants (Li et al., 2014 ). In the present study, higher bacterial alpha-diversity indices were found in healthy plants than in wilt diseased plants, but this is not true for fungal community. Higher diversity in soil bacteria is often associated with greater resistance to pathogens (Garbeva et al., 2004 ; Mallon et al., 2015 ). The increase of soil resistance/tolerance to pathogens may be related to the complexity of the interaction network of microorganisms in soil (Shi et al., 2019 ). Complex microbial community interaction can regulate the stability of the community (Eisenhauer et al., 2013 ), thus limiting pathogen increase. Soilborne pathogens can have profound impacts on nutrient availability and plant root exudates in rhizosphere, which, in turn, may affect microbial communities (Cook et al., 1995 ). The present study showed large and consistent effects of a soilborne vascular disease on both fungal and bacterial communities in the rhizosphere of cotton plants. Rhizosphere microbiomes have been showed to differ between healthy and soilborne diseased plants (Mendes et al., 2011 ; Shanmugam et al., 2011 ; Zhang et al., 2011 ; Wu et al., 2015 ). However, a recent study showed that bacterial community in the geocaulosphere soil could be distinguished according to potato scab severity, but not in rhizosphere soil (Shi et al., 2019 ). This difference could be because that common scab of potato mainly invades through tubers, whereas many other soilborne pathogens directly infect root systems. As a vascular pathogen of the cotton, V. dahliae infects roots and then colonizes vascular tissues, causing plant wilting, but usually does not cause root decay. However, fungal colonization in vascular tissues is expected to result in considerable changes in plant physiology and hence in root exudates. We thus speculate that it is the change in root exudates that may largely be responsible for the resulting differences in rhizosphere microbiomes between healthy and diseased plants. In the present study, the differences in the rhizosphere microbiome between diseased and healthy plants within the same cultivars are much greater than differences between cultivars. Cultivar differences may be exaggerated as the two cultivars were grown separately in two neighboring fields and thus are confounded with the differences in microbial communities between the two fields, which is well-known (Edwards et al., 2015 ). Such a spatial effect was also illustrated by the significant differences between pairs of plants (representing different locations within a field). Precisely for this reason, we sampled scheme neighboring diseased and healthy plants of the same cultivar to minimize the compounding effects of disease phenotype and microbial variability in space. Thus, the differences in the rhizosphere microbial community between diseased and healthy plant within the pairs of plants are more likely to be directly related to the wilt development. The important question, however, remains whether the large community differences in the rhizosphere between diseased and healthy plants are a consequence of the infection by V . dahliae and/or subsequent wilt development that has affected root exudate composition leading to changes in rhizosphere microbiome. Differences in abundance of individual OTUs between the diseased and healthy plants have a consistent pattern: nearly all those bacterial OTUs with differential abundance had much higher abundance in healthy plants than in diseased plants, and the opposite was true for the fungal OTUs. Many bacterial and some fungal groups have been shown to associate with cotton plant tolerance to development of wilt caused by V . dahliae (Wei et al., 2019 ). Many specific bacterial OTUs have higher abundance in rhizosphere of healthy plants than diseased plants. For instance, the abundance of Acidobacteria is reduced in rhizosphere of diseased plants, agreeing with a recent finding that the decrease of this phylum is linked to wilt development in olive, also caused by V. dahliae (Fernández-González et al., 2020 ). Furthermore, the abundance of several well-known taxonomy groups containing beneficial microbes, such as Bacilli (Firmicutes) and Gemmatimonadetes, was reduced in the rhizosphere of diseased plants, consistent with previous findings (Wei et al., 2019 ; Fernández-González et al., 2020 ). In contrast, the abundance of Gammaproteobacteria was increased in diseased plants in both cultivars. Gammaproteobacterial diversity and community members have been identified as potential health indicators (Köberl et al., 2017 ). For example, healthy banana ( Musa acuminata L.) plants have increased presence of potentially plant-beneficial Pseudomonas and Stenotrophomonas , whereas diseased plants had a high level of Enterobacteriaceae known for their plant degradation ability (Köberl et al., 2017 ). Decreased abundance associated with diseased plants was also observed for arbuscular mycorrhizal fungi (AMF). AMF colonization may have increased the expression of pathogenesis-related genes and lignin synthesis-related genes more strongly, thus leading to induced resistance against V . dahliae in cotton (Zhang et al., 2018 ). In addition, AMF can also induce changes in the composition of cotton root exudates, contributing to bioactive effects against germination of V . dahliae conidia (Zhang et al., 2012a ). Similarly, increased abundance of fungal pathogens in general together with reduced abundance of beneficial microbes may explain yield (mainly due to soilborne diseases) declining potential observed in continuous cotton monocropping systems (Wei and Yu, 2018 ). The present study showed that a much greater proportion of rhizosphere microbial OTUs differed in their abundance between neighboring diseased and healthy plants of the same cultivars than between cultivars with differing susceptibility to V . dahliae . This may suggest that much of these differences between diseased and healthy plants may have resulted from postinfection or postdisease development as consequences of changes in root exudates and/or volatiles from plants associated with pathogen infection and subsequent disease development. Thus, rhizosphere of diseased plants has greater abundance of fungal saprophytes than healthy plants."
} | 4,087 |
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